Comparison of RPS15 Expression Levels between Normal and Tumour Cells of Human Nasopharyngeal Epithelium

ONG LIAN WEEN

(43675)

Bachelor of Science with Honours (Resource Biotechnology)

2016

P KHIDKAT KAKLUKAT AKADEKIKPu Akadrmillt U iARAWAllt

11111111111111111111111 1000272661

Comparison of RPS15 Expression Levels between Normal and Tumour Cells of Human Nasopharyngeal Epithelium

ONG LIAN WEEN (43675)

This Final Year Project Report is submitted in partial fulfilment of the requirement for the degree of Bachelor of Science with Honours (Resource Biotechnology)

Supervisor Associate Professor Dr Edmund Sim Vi Hang

Bachelor of Science (Honours) Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

2016

Acknowledgement

This project would not have come to its beautiful ending if there are no assistance and

support from many people all the way through the end of this project I would like to express

my deepest gratitude to Associate Professor Dr Edmund Sim Ui Hang my Final Year

Project supervisor for his patient guidance and for his appreciable and constructive critiques

on this research project I would also like to acknowledge my role model Ms Ng Kher Lee

for her guidance in keeping my progress on schedule Her willingness in sharing her

knowledge so generously has been very much appreciated Furthennore I would also like to

give my special thanks to Ms Felicia Kavita Thomas who is a cell culture expert for her

continuous support in providing me cultures ofnasopharyngeal cell lines My grateful thanks

are also extended to Ms Shruti Talwar for her enthusiastic encouragement Thanks are also

due to Ms Stella Chan Li Li Ms Yew Keh Li and Ms Cassandra Chee Sheau Mei for their

useful recommendations

I would like to extend my thanks to my FYP team mates they are Selvamalar AlP

Mutsamy Nur Atiqah Binti Azman and Teh Zy Ying Our friendships have go fonder as we

went through hardship and happiness together I am particularly grateful for the assistance

from Laboratory Assistance Ms Limjatai Kadin ak Patrick for her help and warmth concern

on my progress

Last but not least I wish to express my warmly and deeply thanks to my parents for

their encouragement and their cares given to me Their concerns on my academic have

providing me a great strength to keep on strives for success in my undergraduate life And

not to forget my sister who has been continually listen to my stories during my delightful

and cheerless moments Finally thanks to my housemates and course mates for their

friendships

I

UNIVERSITI MALAYSIA SARAWAK

Grade

Please tick (J) Final Year Project Report

Masters

PhD

DECLARATION OF ORIGINAL WORK

This declaration is made on the 4-~~day of(1~Y~2016

Students Declaration

lONG LIAN WEEN 43675 Faculty of Resource Science and Technology hereby declare that the work entitled Comparison of RPS15 Expression Levels between Normal and Tumour Cells of Human Nasopharyngeal Epithelium is my original work I have not copied from any other students work or from any other sources except where due reference or acknowledgement is made explicitly in the text nor has any part been written for me by another person

~ ~t4((Date submitted

01Jlty ~ft1iJ WPf4J (~3 b7~) Name of the student (Matric No)

Supervisors Declaration

I__~~~__~_epound__~~~J~~~~Lamp~ __~~__~~j__ (SUPERVISORS NAME) hereby certifies that the work entitled __________~L_~~~+_~~__~__~~_________________________------(TITLE) was prepared

by the above named student and was submitted tathe FAClILTY as a partialfull fulfillment for the conferment of _____~_~~~_~~_l----~~_~~ __~~fd~~~~)---------- (PLEASE INDICATE THE DEGREE) and the aforementioned work to the best of my knowledge is the

said students work

Received for examination by ~t ft tgtr ~VIoc (Name of the supervisor)

II

I declare that ProjectThesis is classified as (Please tick (Jraquo

D CONFIDENTIAL (Contains confidential information under the Official Secret Act 1972) DRESTRICTED (Contains restricted information as specified by the organization where

research was done) ~OPEN ACCESS

Validation of ProjectThesis

I therefore duly affirm with free consent and willingly declare that this said ProjectlThesis shall be placed officially in the Centre for Academic Information Services with the abiding interest and rights as follows

bull This ProjectlThesis IS the sole legal property of Universiti Malaysia Sarawak (UNlMAS)

bull The Centre for Academic Information Services has the lawful right to make copies for the purpose of academic and research only and not for other purpose

bull The Centre for Academic Information Services has the lawful right to digitalize the content for the Local Content Database

bull The Centre for Academic Information Services has the lawful right to make copies of the ProjectThesis for academic exchange between Higher Learning Institute

bull No dispute or any claim shall arise from the student itself neither third party on this ProjectlThesis once it becomes the sole property of UNlMAS

bull This ProjectlThesis or any material data and information related to it shall not be distributed published or disclosed to any party by the student except with UNlMAS permISSIon

-

~ Student signature llL 2 21tltJl Supervisor signature L-a middot _ shy

(Date) (Date)

Current Address 6 TAMAN MA WAR JALAN MA WAR

06700 PENDANG KEDAH DARUL AMAN

Notes If the ProjectThesis is CONFIDENTIAL or RESTRICTED please attach together as annexure a letter from the organization with the period and reasons of confidentiality and restriction

[The instrument is duly prepared by The Centre for Academic Information Services]

III

Pusat Khidmat Maklumat Akad~mi~ UNlVERSm MALAYSIA SARAWAt~

Table of Content

Contents Page Title and Front Cover Acknowledgement I Declaration II Content Page IV List of Abbreviations VI List of Tables VII List of Figures VIII Abstract in English and Bahasa Malaysia 1 10 Introduction 2

11 Background 2 12 Objectives 3

20 Literature Review 4 21 Nasopharyngeal Carcinoma (NPC) 4

211 Causative Factors ofNPC 4 212 Staging ofNPC 5 213 Histopathological types ofNPC 5 214 Genes Linked to NPC 6

22 Ribosomal Proteins (RPs) 7 221 Extra-ribosomal functions ofRPs 7

23 Ribosomal Protein S 15 (RPS 15) 8 231 The Relevance ofRPS15 with Various Hematologic

Disorders 9 2311 Mutation ofRPs caused ribosomopathies 9 2312 Interaction ofRPs with p53 9

24 Cell Lines Used in RPS 15 Gene Expression Level Analysis 10 241 NP69 cell line 10 242 NPCHKl cell line I t

25 Analysis of Gene Expression 11 251 Housekeeping gene as a control 11

2511 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene 12

26 Principles of Methodology 12 261 The Advantages of TRIzol Reagent 12 262 Qualitative and Quantitative Analysis of RNA 13 263 Considerations on Designing Primer 14 264 Optimization of Primer Annealing Temperature 15 265 Direct Sequencing ofPCR Products 15 266 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 16

IV

30 Methodology 18 31 RNA Extraction 18 32 RNA Confinnation Steps 19

321 Quantifying the Concentration of RNA 19 322 Checking RNA Quality using Agarose Gel Electrophoresis 20

(AGE) 33 Reverse Transcription of RNA sample 20 34 Designing Primer Sets for GAPDH and RPSJ5 21 35 Gradient PCR for Optimization of Primer Annealing Temperature 22 36 Sequencing of Amplified PCR Products 24 37 Optimized RT-PCR for Gene Expression Analysis 25 38 Analysis of Band Intensities 27

40 Results 28 41 Qualitative and Quantitative Analysis of RNA Samples 28 42 Gradient PCR for Optimization on the Annealing Temperature of 29GAPDH and RPSJ5 43 The Identity of Amplified Sequences 31 44 Optimized RT-PCR for Analysis of Gene Expression Levels 32 45 Statistical Data Analysis 33

50 Discussion 38 51 The Possible Effects on Overexpression ofRP Gene 41 52 Limitations ofRT-PCR 42

60 Conclusion 44 61 Recommendations 44

70 References 45

Appendices 50 Appendix 1 Various Requirements for Primer Design 50 Appendix 2 Sequencing Results for RPSJ5 and GAPDH 54 Appendix 3 Band Intensities Analysis 64 Appendix 4 Statistical Data Analysis 65

V

1 1

List of Abbreviations

A260

A280

AGE Cdk eDNA eLL Co-IP DBA BBV EtBr GAPDH HCC HLA ICAT LMPI MALDI-TOF Mdm2 M-MLVRT NOMA NP NPC NPIP NPYR NTC PCR PTEN RING RNA RP RPS15 rRNA RT-PCR SCC Spl Ta TAE Taopi Tm UV-spec UV-trans WHO

Absorbance at 260nm Absorbance at 280nm Agarose Ge1 Electrophoresis CyeliD-dependent kinase complementary DNA Chronic lymphocytic leukemia Co-Immunoprecipitation Dark-Blackfan Anaemia Epstein-Barr Virus Ethidium Bromide Glyceraldehyde-3-Phosphate Dehydrogenase Human Hepatocellular Carcinoma Human Leukocyte Antigen Isotope-coded Affinity Tag EBV -encoded latent membrane protein 1 Matrix-Assisted Laser Desorption Ionization with Time-of-Flight Mouse double minute 2 Moloney Murine Leukemia Virus Reverse Transcriptase N i trosodimethyamine Nasopharyngeal Nasopharyngeal Carcinoma N -nitrospiperidine N -nitrospyrrolidene Non-template control Polymerase Chain Reaction Phosphatase and tensin homolog Really Interesting New Gene Ribonueleic Acid Ribosomal Protein Ribosomal Protein S 15 ribosomal RNA Reverse Transcription Polymerase Chain Reaction Squamous Cell Carcinoma Specific protein 1 Annealing Temperature Tris-Acetate-Ethylenediamine-Tetraacetic-Acid Optimum Annealing Temperature melting temperature UV-spectrophotometer UV transilluminator World Health Organization

VI

r

List of Tables

Table Page 31 The components that were added into the 151lL mixture of RNA sample 21

random primers and nuclease free water

32 The PCR Master Mix that was prepared for 7 reactions to test for Ta of 23 RPS15 primer set

33 The PCR Master Mix that was prepared for 5 reactions to test for Ta of 23 GAPDH primer set

34 The generalized representation for the components of test reactions and 23 negative control reactions

35 The parameters of PCR reaction for RPS15 24

36 The parameters ofPCR reaction for GAPDH 24

37 The components of the PCR Master Mix 26

38 The components ofthe test reaction and positive as well as negative control 26 reactions that were added to PCR Master Mix

39 The PCR profile of the Optimized PCR for all the reactions 26

41 The absorbance readings concentrations of RNA and absorbance ratios 29 for all the RNA extracted from NP69 and HKl

42 Band intensities of all the genes based on pixel volumes quantified using 34 ImageQuant TL Software

43 Nonnalized RPS15 values 36

VII

List of Figures

Figure Page Circled indicates the location ofRPS15 gene in chromosome 19 ofHomo

21 sapiens (Adapted from httpwwwncbinlmnihgovgene6209) 8

RNA Integrity Checking through AGE (A) The gel image of intact fresh RNA extracted from HKl as viewed on UV-transilluminator (B) The gel

41 image of intact RNA extracted from NP69 captured by iQuant Capture 28 Software

Amplification of GAPDH using gradient PCR to detennine the T aOpt

42 (Lane 1=493degC Lane 2=507degC Lane 3=530oC) 30

Amplification ofRPS15 using gradient PCR to detennine the T aOpt (Lane 1=570oC Lane 2=576degC Lane 3=604degC Lane 4=642degC Lane43 305=660oC)

Analysis of expression of RPS15 in nonnal and tumour nasopharyngeal 44 epithelium cell lines with the positive control GAPDHusing RT-PCR 33

Graphical representation on the pixel volumes of each genes in each cell lines plotted together with the mean and error bars from the average of45 35the readings of each genes in each cell lines

Average of the nonnalized RPS15 values in NP69 nonnal cell line and HK1 tumour cell line NP69 plot n=2 HKl plot n=2 Two sample 46 36Unpaired I-test 2 degree of freedom 95 confident level plt005

VIII

P KHIDKAT KAKLUKAT AKADEKIKPu Akadrmillt U iARAWAllt

11111111111111111111111 1000272661

Comparison of RPS15 Expression Levels between Normal and Tumour Cells of Human Nasopharyngeal Epithelium

ONG LIAN WEEN (43675)

This Final Year Project Report is submitted in partial fulfilment of the requirement for the degree of Bachelor of Science with Honours (Resource Biotechnology)

Supervisor Associate Professor Dr Edmund Sim Vi Hang

Bachelor of Science (Honours) Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

2016

Acknowledgement

This project would not have come to its beautiful ending if there are no assistance and

support from many people all the way through the end of this project I would like to express

my deepest gratitude to Associate Professor Dr Edmund Sim Ui Hang my Final Year

Project supervisor for his patient guidance and for his appreciable and constructive critiques

on this research project I would also like to acknowledge my role model Ms Ng Kher Lee

for her guidance in keeping my progress on schedule Her willingness in sharing her

knowledge so generously has been very much appreciated Furthennore I would also like to

give my special thanks to Ms Felicia Kavita Thomas who is a cell culture expert for her

continuous support in providing me cultures ofnasopharyngeal cell lines My grateful thanks

are also extended to Ms Shruti Talwar for her enthusiastic encouragement Thanks are also

due to Ms Stella Chan Li Li Ms Yew Keh Li and Ms Cassandra Chee Sheau Mei for their

useful recommendations

I would like to extend my thanks to my FYP team mates they are Selvamalar AlP

Mutsamy Nur Atiqah Binti Azman and Teh Zy Ying Our friendships have go fonder as we

went through hardship and happiness together I am particularly grateful for the assistance

from Laboratory Assistance Ms Limjatai Kadin ak Patrick for her help and warmth concern

on my progress

Last but not least I wish to express my warmly and deeply thanks to my parents for

their encouragement and their cares given to me Their concerns on my academic have

providing me a great strength to keep on strives for success in my undergraduate life And

not to forget my sister who has been continually listen to my stories during my delightful

and cheerless moments Finally thanks to my housemates and course mates for their

friendships

I

UNIVERSITI MALAYSIA SARAWAK

Grade

Please tick (J) Final Year Project Report

Masters

PhD

DECLARATION OF ORIGINAL WORK

This declaration is made on the 4-~~day of(1~Y~2016

Students Declaration

lONG LIAN WEEN 43675 Faculty of Resource Science and Technology hereby declare that the work entitled Comparison of RPS15 Expression Levels between Normal and Tumour Cells of Human Nasopharyngeal Epithelium is my original work I have not copied from any other students work or from any other sources except where due reference or acknowledgement is made explicitly in the text nor has any part been written for me by another person

~ ~t4((Date submitted

01Jlty ~ft1iJ WPf4J (~3 b7~) Name of the student (Matric No)

Supervisors Declaration

I__~~~__~_epound__~~~J~~~~Lamp~ __~~__~~j__ (SUPERVISORS NAME) hereby certifies that the work entitled __________~L_~~~+_~~__~__~~_________________________------(TITLE) was prepared

by the above named student and was submitted tathe FAClILTY as a partialfull fulfillment for the conferment of _____~_~~~_~~_l----~~_~~ __~~fd~~~~)---------- (PLEASE INDICATE THE DEGREE) and the aforementioned work to the best of my knowledge is the

said students work

Received for examination by ~t ft tgtr ~VIoc (Name of the supervisor)

II

I declare that ProjectThesis is classified as (Please tick (Jraquo

D CONFIDENTIAL (Contains confidential information under the Official Secret Act 1972) DRESTRICTED (Contains restricted information as specified by the organization where

research was done) ~OPEN ACCESS

Validation of ProjectThesis

I therefore duly affirm with free consent and willingly declare that this said ProjectlThesis shall be placed officially in the Centre for Academic Information Services with the abiding interest and rights as follows

bull This ProjectlThesis IS the sole legal property of Universiti Malaysia Sarawak (UNlMAS)

bull The Centre for Academic Information Services has the lawful right to make copies for the purpose of academic and research only and not for other purpose

bull The Centre for Academic Information Services has the lawful right to digitalize the content for the Local Content Database

bull The Centre for Academic Information Services has the lawful right to make copies of the ProjectThesis for academic exchange between Higher Learning Institute

bull No dispute or any claim shall arise from the student itself neither third party on this ProjectlThesis once it becomes the sole property of UNlMAS

bull This ProjectlThesis or any material data and information related to it shall not be distributed published or disclosed to any party by the student except with UNlMAS permISSIon

-

~ Student signature llL 2 21tltJl Supervisor signature L-a middot _ shy

(Date) (Date)

Current Address 6 TAMAN MA WAR JALAN MA WAR

06700 PENDANG KEDAH DARUL AMAN

Notes If the ProjectThesis is CONFIDENTIAL or RESTRICTED please attach together as annexure a letter from the organization with the period and reasons of confidentiality and restriction

[The instrument is duly prepared by The Centre for Academic Information Services]

III

Pusat Khidmat Maklumat Akad~mi~ UNlVERSm MALAYSIA SARAWAt~

Table of Content

Contents Page Title and Front Cover Acknowledgement I Declaration II Content Page IV List of Abbreviations VI List of Tables VII List of Figures VIII Abstract in English and Bahasa Malaysia 1 10 Introduction 2

11 Background 2 12 Objectives 3

20 Literature Review 4 21 Nasopharyngeal Carcinoma (NPC) 4

211 Causative Factors ofNPC 4 212 Staging ofNPC 5 213 Histopathological types ofNPC 5 214 Genes Linked to NPC 6

22 Ribosomal Proteins (RPs) 7 221 Extra-ribosomal functions ofRPs 7

23 Ribosomal Protein S 15 (RPS 15) 8 231 The Relevance ofRPS15 with Various Hematologic

Disorders 9 2311 Mutation ofRPs caused ribosomopathies 9 2312 Interaction ofRPs with p53 9

24 Cell Lines Used in RPS 15 Gene Expression Level Analysis 10 241 NP69 cell line 10 242 NPCHKl cell line I t

25 Analysis of Gene Expression 11 251 Housekeeping gene as a control 11

2511 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene 12

26 Principles of Methodology 12 261 The Advantages of TRIzol Reagent 12 262 Qualitative and Quantitative Analysis of RNA 13 263 Considerations on Designing Primer 14 264 Optimization of Primer Annealing Temperature 15 265 Direct Sequencing ofPCR Products 15 266 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 16

IV

30 Methodology 18 31 RNA Extraction 18 32 RNA Confinnation Steps 19

321 Quantifying the Concentration of RNA 19 322 Checking RNA Quality using Agarose Gel Electrophoresis 20

(AGE) 33 Reverse Transcription of RNA sample 20 34 Designing Primer Sets for GAPDH and RPSJ5 21 35 Gradient PCR for Optimization of Primer Annealing Temperature 22 36 Sequencing of Amplified PCR Products 24 37 Optimized RT-PCR for Gene Expression Analysis 25 38 Analysis of Band Intensities 27

40 Results 28 41 Qualitative and Quantitative Analysis of RNA Samples 28 42 Gradient PCR for Optimization on the Annealing Temperature of 29GAPDH and RPSJ5 43 The Identity of Amplified Sequences 31 44 Optimized RT-PCR for Analysis of Gene Expression Levels 32 45 Statistical Data Analysis 33

50 Discussion 38 51 The Possible Effects on Overexpression ofRP Gene 41 52 Limitations ofRT-PCR 42

60 Conclusion 44 61 Recommendations 44

70 References 45

Appendices 50 Appendix 1 Various Requirements for Primer Design 50 Appendix 2 Sequencing Results for RPSJ5 and GAPDH 54 Appendix 3 Band Intensities Analysis 64 Appendix 4 Statistical Data Analysis 65

V

1 1

List of Abbreviations

A260

A280

AGE Cdk eDNA eLL Co-IP DBA BBV EtBr GAPDH HCC HLA ICAT LMPI MALDI-TOF Mdm2 M-MLVRT NOMA NP NPC NPIP NPYR NTC PCR PTEN RING RNA RP RPS15 rRNA RT-PCR SCC Spl Ta TAE Taopi Tm UV-spec UV-trans WHO

Absorbance at 260nm Absorbance at 280nm Agarose Ge1 Electrophoresis CyeliD-dependent kinase complementary DNA Chronic lymphocytic leukemia Co-Immunoprecipitation Dark-Blackfan Anaemia Epstein-Barr Virus Ethidium Bromide Glyceraldehyde-3-Phosphate Dehydrogenase Human Hepatocellular Carcinoma Human Leukocyte Antigen Isotope-coded Affinity Tag EBV -encoded latent membrane protein 1 Matrix-Assisted Laser Desorption Ionization with Time-of-Flight Mouse double minute 2 Moloney Murine Leukemia Virus Reverse Transcriptase N i trosodimethyamine Nasopharyngeal Nasopharyngeal Carcinoma N -nitrospiperidine N -nitrospyrrolidene Non-template control Polymerase Chain Reaction Phosphatase and tensin homolog Really Interesting New Gene Ribonueleic Acid Ribosomal Protein Ribosomal Protein S 15 ribosomal RNA Reverse Transcription Polymerase Chain Reaction Squamous Cell Carcinoma Specific protein 1 Annealing Temperature Tris-Acetate-Ethylenediamine-Tetraacetic-Acid Optimum Annealing Temperature melting temperature UV-spectrophotometer UV transilluminator World Health Organization

VI

r

List of Tables

Table Page 31 The components that were added into the 151lL mixture of RNA sample 21

random primers and nuclease free water

32 The PCR Master Mix that was prepared for 7 reactions to test for Ta of 23 RPS15 primer set

33 The PCR Master Mix that was prepared for 5 reactions to test for Ta of 23 GAPDH primer set

34 The generalized representation for the components of test reactions and 23 negative control reactions

35 The parameters of PCR reaction for RPS15 24

36 The parameters ofPCR reaction for GAPDH 24

37 The components of the PCR Master Mix 26

38 The components ofthe test reaction and positive as well as negative control 26 reactions that were added to PCR Master Mix

39 The PCR profile of the Optimized PCR for all the reactions 26

41 The absorbance readings concentrations of RNA and absorbance ratios 29 for all the RNA extracted from NP69 and HKl

42 Band intensities of all the genes based on pixel volumes quantified using 34 ImageQuant TL Software

43 Nonnalized RPS15 values 36

VII

List of Figures

Figure Page Circled indicates the location ofRPS15 gene in chromosome 19 ofHomo

21 sapiens (Adapted from httpwwwncbinlmnihgovgene6209) 8

RNA Integrity Checking through AGE (A) The gel image of intact fresh RNA extracted from HKl as viewed on UV-transilluminator (B) The gel

41 image of intact RNA extracted from NP69 captured by iQuant Capture 28 Software

Amplification of GAPDH using gradient PCR to detennine the T aOpt

42 (Lane 1=493degC Lane 2=507degC Lane 3=530oC) 30

Amplification ofRPS15 using gradient PCR to detennine the T aOpt (Lane 1=570oC Lane 2=576degC Lane 3=604degC Lane 4=642degC Lane43 305=660oC)

Analysis of expression of RPS15 in nonnal and tumour nasopharyngeal 44 epithelium cell lines with the positive control GAPDHusing RT-PCR 33

Graphical representation on the pixel volumes of each genes in each cell lines plotted together with the mean and error bars from the average of45 35the readings of each genes in each cell lines

Average of the nonnalized RPS15 values in NP69 nonnal cell line and HK1 tumour cell line NP69 plot n=2 HKl plot n=2 Two sample 46 36Unpaired I-test 2 degree of freedom 95 confident level plt005

VIII

Acknowledgement

This project would not have come to its beautiful ending if there are no assistance and

support from many people all the way through the end of this project I would like to express

my deepest gratitude to Associate Professor Dr Edmund Sim Ui Hang my Final Year

Project supervisor for his patient guidance and for his appreciable and constructive critiques

on this research project I would also like to acknowledge my role model Ms Ng Kher Lee

for her guidance in keeping my progress on schedule Her willingness in sharing her

knowledge so generously has been very much appreciated Furthennore I would also like to

give my special thanks to Ms Felicia Kavita Thomas who is a cell culture expert for her

continuous support in providing me cultures ofnasopharyngeal cell lines My grateful thanks

are also extended to Ms Shruti Talwar for her enthusiastic encouragement Thanks are also

due to Ms Stella Chan Li Li Ms Yew Keh Li and Ms Cassandra Chee Sheau Mei for their

useful recommendations

I would like to extend my thanks to my FYP team mates they are Selvamalar AlP

Mutsamy Nur Atiqah Binti Azman and Teh Zy Ying Our friendships have go fonder as we

went through hardship and happiness together I am particularly grateful for the assistance

from Laboratory Assistance Ms Limjatai Kadin ak Patrick for her help and warmth concern

on my progress

Last but not least I wish to express my warmly and deeply thanks to my parents for

their encouragement and their cares given to me Their concerns on my academic have

providing me a great strength to keep on strives for success in my undergraduate life And

not to forget my sister who has been continually listen to my stories during my delightful

and cheerless moments Finally thanks to my housemates and course mates for their

friendships

I

UNIVERSITI MALAYSIA SARAWAK

Grade

Please tick (J) Final Year Project Report

Masters

PhD

DECLARATION OF ORIGINAL WORK

This declaration is made on the 4-~~day of(1~Y~2016

Students Declaration

lONG LIAN WEEN 43675 Faculty of Resource Science and Technology hereby declare that the work entitled Comparison of RPS15 Expression Levels between Normal and Tumour Cells of Human Nasopharyngeal Epithelium is my original work I have not copied from any other students work or from any other sources except where due reference or acknowledgement is made explicitly in the text nor has any part been written for me by another person

~ ~t4((Date submitted

01Jlty ~ft1iJ WPf4J (~3 b7~) Name of the student (Matric No)

Supervisors Declaration

I__~~~__~_epound__~~~J~~~~Lamp~ __~~__~~j__ (SUPERVISORS NAME) hereby certifies that the work entitled __________~L_~~~+_~~__~__~~_________________________------(TITLE) was prepared

by the above named student and was submitted tathe FAClILTY as a partialfull fulfillment for the conferment of _____~_~~~_~~_l----~~_~~ __~~fd~~~~)---------- (PLEASE INDICATE THE DEGREE) and the aforementioned work to the best of my knowledge is the

said students work

Received for examination by ~t ft tgtr ~VIoc (Name of the supervisor)

II

I declare that ProjectThesis is classified as (Please tick (Jraquo

D CONFIDENTIAL (Contains confidential information under the Official Secret Act 1972) DRESTRICTED (Contains restricted information as specified by the organization where

research was done) ~OPEN ACCESS

Validation of ProjectThesis

I therefore duly affirm with free consent and willingly declare that this said ProjectlThesis shall be placed officially in the Centre for Academic Information Services with the abiding interest and rights as follows

bull This ProjectlThesis IS the sole legal property of Universiti Malaysia Sarawak (UNlMAS)

bull The Centre for Academic Information Services has the lawful right to make copies for the purpose of academic and research only and not for other purpose

bull The Centre for Academic Information Services has the lawful right to digitalize the content for the Local Content Database

bull The Centre for Academic Information Services has the lawful right to make copies of the ProjectThesis for academic exchange between Higher Learning Institute

bull No dispute or any claim shall arise from the student itself neither third party on this ProjectlThesis once it becomes the sole property of UNlMAS

bull This ProjectlThesis or any material data and information related to it shall not be distributed published or disclosed to any party by the student except with UNlMAS permISSIon

-

~ Student signature llL 2 21tltJl Supervisor signature L-a middot _ shy

(Date) (Date)

Current Address 6 TAMAN MA WAR JALAN MA WAR

06700 PENDANG KEDAH DARUL AMAN

Notes If the ProjectThesis is CONFIDENTIAL or RESTRICTED please attach together as annexure a letter from the organization with the period and reasons of confidentiality and restriction

[The instrument is duly prepared by The Centre for Academic Information Services]

III

Pusat Khidmat Maklumat Akad~mi~ UNlVERSm MALAYSIA SARAWAt~

Table of Content

Contents Page Title and Front Cover Acknowledgement I Declaration II Content Page IV List of Abbreviations VI List of Tables VII List of Figures VIII Abstract in English and Bahasa Malaysia 1 10 Introduction 2

11 Background 2 12 Objectives 3

20 Literature Review 4 21 Nasopharyngeal Carcinoma (NPC) 4

211 Causative Factors ofNPC 4 212 Staging ofNPC 5 213 Histopathological types ofNPC 5 214 Genes Linked to NPC 6

22 Ribosomal Proteins (RPs) 7 221 Extra-ribosomal functions ofRPs 7

23 Ribosomal Protein S 15 (RPS 15) 8 231 The Relevance ofRPS15 with Various Hematologic

Disorders 9 2311 Mutation ofRPs caused ribosomopathies 9 2312 Interaction ofRPs with p53 9

24 Cell Lines Used in RPS 15 Gene Expression Level Analysis 10 241 NP69 cell line 10 242 NPCHKl cell line I t

25 Analysis of Gene Expression 11 251 Housekeeping gene as a control 11

2511 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene 12

26 Principles of Methodology 12 261 The Advantages of TRIzol Reagent 12 262 Qualitative and Quantitative Analysis of RNA 13 263 Considerations on Designing Primer 14 264 Optimization of Primer Annealing Temperature 15 265 Direct Sequencing ofPCR Products 15 266 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 16

IV

30 Methodology 18 31 RNA Extraction 18 32 RNA Confinnation Steps 19

321 Quantifying the Concentration of RNA 19 322 Checking RNA Quality using Agarose Gel Electrophoresis 20

(AGE) 33 Reverse Transcription of RNA sample 20 34 Designing Primer Sets for GAPDH and RPSJ5 21 35 Gradient PCR for Optimization of Primer Annealing Temperature 22 36 Sequencing of Amplified PCR Products 24 37 Optimized RT-PCR for Gene Expression Analysis 25 38 Analysis of Band Intensities 27

40 Results 28 41 Qualitative and Quantitative Analysis of RNA Samples 28 42 Gradient PCR for Optimization on the Annealing Temperature of 29GAPDH and RPSJ5 43 The Identity of Amplified Sequences 31 44 Optimized RT-PCR for Analysis of Gene Expression Levels 32 45 Statistical Data Analysis 33

50 Discussion 38 51 The Possible Effects on Overexpression ofRP Gene 41 52 Limitations ofRT-PCR 42

60 Conclusion 44 61 Recommendations 44

70 References 45

Appendices 50 Appendix 1 Various Requirements for Primer Design 50 Appendix 2 Sequencing Results for RPSJ5 and GAPDH 54 Appendix 3 Band Intensities Analysis 64 Appendix 4 Statistical Data Analysis 65

V

1 1

List of Abbreviations

A260

A280

AGE Cdk eDNA eLL Co-IP DBA BBV EtBr GAPDH HCC HLA ICAT LMPI MALDI-TOF Mdm2 M-MLVRT NOMA NP NPC NPIP NPYR NTC PCR PTEN RING RNA RP RPS15 rRNA RT-PCR SCC Spl Ta TAE Taopi Tm UV-spec UV-trans WHO

Absorbance at 260nm Absorbance at 280nm Agarose Ge1 Electrophoresis CyeliD-dependent kinase complementary DNA Chronic lymphocytic leukemia Co-Immunoprecipitation Dark-Blackfan Anaemia Epstein-Barr Virus Ethidium Bromide Glyceraldehyde-3-Phosphate Dehydrogenase Human Hepatocellular Carcinoma Human Leukocyte Antigen Isotope-coded Affinity Tag EBV -encoded latent membrane protein 1 Matrix-Assisted Laser Desorption Ionization with Time-of-Flight Mouse double minute 2 Moloney Murine Leukemia Virus Reverse Transcriptase N i trosodimethyamine Nasopharyngeal Nasopharyngeal Carcinoma N -nitrospiperidine N -nitrospyrrolidene Non-template control Polymerase Chain Reaction Phosphatase and tensin homolog Really Interesting New Gene Ribonueleic Acid Ribosomal Protein Ribosomal Protein S 15 ribosomal RNA Reverse Transcription Polymerase Chain Reaction Squamous Cell Carcinoma Specific protein 1 Annealing Temperature Tris-Acetate-Ethylenediamine-Tetraacetic-Acid Optimum Annealing Temperature melting temperature UV-spectrophotometer UV transilluminator World Health Organization

VI

r

List of Tables

Table Page 31 The components that were added into the 151lL mixture of RNA sample 21

random primers and nuclease free water

32 The PCR Master Mix that was prepared for 7 reactions to test for Ta of 23 RPS15 primer set

33 The PCR Master Mix that was prepared for 5 reactions to test for Ta of 23 GAPDH primer set

34 The generalized representation for the components of test reactions and 23 negative control reactions

35 The parameters of PCR reaction for RPS15 24

36 The parameters ofPCR reaction for GAPDH 24

37 The components of the PCR Master Mix 26

38 The components ofthe test reaction and positive as well as negative control 26 reactions that were added to PCR Master Mix

39 The PCR profile of the Optimized PCR for all the reactions 26

41 The absorbance readings concentrations of RNA and absorbance ratios 29 for all the RNA extracted from NP69 and HKl

42 Band intensities of all the genes based on pixel volumes quantified using 34 ImageQuant TL Software

43 Nonnalized RPS15 values 36

VII

List of Figures

Figure Page Circled indicates the location ofRPS15 gene in chromosome 19 ofHomo

21 sapiens (Adapted from httpwwwncbinlmnihgovgene6209) 8

RNA Integrity Checking through AGE (A) The gel image of intact fresh RNA extracted from HKl as viewed on UV-transilluminator (B) The gel

41 image of intact RNA extracted from NP69 captured by iQuant Capture 28 Software

Amplification of GAPDH using gradient PCR to detennine the T aOpt

42 (Lane 1=493degC Lane 2=507degC Lane 3=530oC) 30

Amplification ofRPS15 using gradient PCR to detennine the T aOpt (Lane 1=570oC Lane 2=576degC Lane 3=604degC Lane 4=642degC Lane43 305=660oC)

Analysis of expression of RPS15 in nonnal and tumour nasopharyngeal 44 epithelium cell lines with the positive control GAPDHusing RT-PCR 33

Graphical representation on the pixel volumes of each genes in each cell lines plotted together with the mean and error bars from the average of45 35the readings of each genes in each cell lines

Average of the nonnalized RPS15 values in NP69 nonnal cell line and HK1 tumour cell line NP69 plot n=2 HKl plot n=2 Two sample 46 36Unpaired I-test 2 degree of freedom 95 confident level plt005

VIII

UNIVERSITI MALAYSIA SARAWAK

Grade

Please tick (J) Final Year Project Report

Masters

PhD

DECLARATION OF ORIGINAL WORK

This declaration is made on the 4-~~day of(1~Y~2016

Students Declaration

lONG LIAN WEEN 43675 Faculty of Resource Science and Technology hereby declare that the work entitled Comparison of RPS15 Expression Levels between Normal and Tumour Cells of Human Nasopharyngeal Epithelium is my original work I have not copied from any other students work or from any other sources except where due reference or acknowledgement is made explicitly in the text nor has any part been written for me by another person

~ ~t4((Date submitted

01Jlty ~ft1iJ WPf4J (~3 b7~) Name of the student (Matric No)

Supervisors Declaration

I__~~~__~_epound__~~~J~~~~Lamp~ __~~__~~j__ (SUPERVISORS NAME) hereby certifies that the work entitled __________~L_~~~+_~~__~__~~_________________________------(TITLE) was prepared

by the above named student and was submitted tathe FAClILTY as a partialfull fulfillment for the conferment of _____~_~~~_~~_l----~~_~~ __~~fd~~~~)---------- (PLEASE INDICATE THE DEGREE) and the aforementioned work to the best of my knowledge is the

said students work

Received for examination by ~t ft tgtr ~VIoc (Name of the supervisor)

II

I declare that ProjectThesis is classified as (Please tick (Jraquo

D CONFIDENTIAL (Contains confidential information under the Official Secret Act 1972) DRESTRICTED (Contains restricted information as specified by the organization where

research was done) ~OPEN ACCESS

Validation of ProjectThesis

I therefore duly affirm with free consent and willingly declare that this said ProjectlThesis shall be placed officially in the Centre for Academic Information Services with the abiding interest and rights as follows

bull This ProjectlThesis IS the sole legal property of Universiti Malaysia Sarawak (UNlMAS)

bull The Centre for Academic Information Services has the lawful right to make copies for the purpose of academic and research only and not for other purpose

bull The Centre for Academic Information Services has the lawful right to digitalize the content for the Local Content Database

bull The Centre for Academic Information Services has the lawful right to make copies of the ProjectThesis for academic exchange between Higher Learning Institute

bull No dispute or any claim shall arise from the student itself neither third party on this ProjectlThesis once it becomes the sole property of UNlMAS

bull This ProjectlThesis or any material data and information related to it shall not be distributed published or disclosed to any party by the student except with UNlMAS permISSIon

-

~ Student signature llL 2 21tltJl Supervisor signature L-a middot _ shy

(Date) (Date)

Current Address 6 TAMAN MA WAR JALAN MA WAR

06700 PENDANG KEDAH DARUL AMAN

Notes If the ProjectThesis is CONFIDENTIAL or RESTRICTED please attach together as annexure a letter from the organization with the period and reasons of confidentiality and restriction

[The instrument is duly prepared by The Centre for Academic Information Services]

III

Pusat Khidmat Maklumat Akad~mi~ UNlVERSm MALAYSIA SARAWAt~

Table of Content

Contents Page Title and Front Cover Acknowledgement I Declaration II Content Page IV List of Abbreviations VI List of Tables VII List of Figures VIII Abstract in English and Bahasa Malaysia 1 10 Introduction 2

11 Background 2 12 Objectives 3

20 Literature Review 4 21 Nasopharyngeal Carcinoma (NPC) 4

211 Causative Factors ofNPC 4 212 Staging ofNPC 5 213 Histopathological types ofNPC 5 214 Genes Linked to NPC 6

22 Ribosomal Proteins (RPs) 7 221 Extra-ribosomal functions ofRPs 7

23 Ribosomal Protein S 15 (RPS 15) 8 231 The Relevance ofRPS15 with Various Hematologic

Disorders 9 2311 Mutation ofRPs caused ribosomopathies 9 2312 Interaction ofRPs with p53 9

24 Cell Lines Used in RPS 15 Gene Expression Level Analysis 10 241 NP69 cell line 10 242 NPCHKl cell line I t

25 Analysis of Gene Expression 11 251 Housekeeping gene as a control 11

2511 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene 12

26 Principles of Methodology 12 261 The Advantages of TRIzol Reagent 12 262 Qualitative and Quantitative Analysis of RNA 13 263 Considerations on Designing Primer 14 264 Optimization of Primer Annealing Temperature 15 265 Direct Sequencing ofPCR Products 15 266 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 16

IV

30 Methodology 18 31 RNA Extraction 18 32 RNA Confinnation Steps 19

321 Quantifying the Concentration of RNA 19 322 Checking RNA Quality using Agarose Gel Electrophoresis 20

(AGE) 33 Reverse Transcription of RNA sample 20 34 Designing Primer Sets for GAPDH and RPSJ5 21 35 Gradient PCR for Optimization of Primer Annealing Temperature 22 36 Sequencing of Amplified PCR Products 24 37 Optimized RT-PCR for Gene Expression Analysis 25 38 Analysis of Band Intensities 27

40 Results 28 41 Qualitative and Quantitative Analysis of RNA Samples 28 42 Gradient PCR for Optimization on the Annealing Temperature of 29GAPDH and RPSJ5 43 The Identity of Amplified Sequences 31 44 Optimized RT-PCR for Analysis of Gene Expression Levels 32 45 Statistical Data Analysis 33

50 Discussion 38 51 The Possible Effects on Overexpression ofRP Gene 41 52 Limitations ofRT-PCR 42

60 Conclusion 44 61 Recommendations 44

70 References 45

Appendices 50 Appendix 1 Various Requirements for Primer Design 50 Appendix 2 Sequencing Results for RPSJ5 and GAPDH 54 Appendix 3 Band Intensities Analysis 64 Appendix 4 Statistical Data Analysis 65

V

1 1

List of Abbreviations

A260

A280

AGE Cdk eDNA eLL Co-IP DBA BBV EtBr GAPDH HCC HLA ICAT LMPI MALDI-TOF Mdm2 M-MLVRT NOMA NP NPC NPIP NPYR NTC PCR PTEN RING RNA RP RPS15 rRNA RT-PCR SCC Spl Ta TAE Taopi Tm UV-spec UV-trans WHO

Absorbance at 260nm Absorbance at 280nm Agarose Ge1 Electrophoresis CyeliD-dependent kinase complementary DNA Chronic lymphocytic leukemia Co-Immunoprecipitation Dark-Blackfan Anaemia Epstein-Barr Virus Ethidium Bromide Glyceraldehyde-3-Phosphate Dehydrogenase Human Hepatocellular Carcinoma Human Leukocyte Antigen Isotope-coded Affinity Tag EBV -encoded latent membrane protein 1 Matrix-Assisted Laser Desorption Ionization with Time-of-Flight Mouse double minute 2 Moloney Murine Leukemia Virus Reverse Transcriptase N i trosodimethyamine Nasopharyngeal Nasopharyngeal Carcinoma N -nitrospiperidine N -nitrospyrrolidene Non-template control Polymerase Chain Reaction Phosphatase and tensin homolog Really Interesting New Gene Ribonueleic Acid Ribosomal Protein Ribosomal Protein S 15 ribosomal RNA Reverse Transcription Polymerase Chain Reaction Squamous Cell Carcinoma Specific protein 1 Annealing Temperature Tris-Acetate-Ethylenediamine-Tetraacetic-Acid Optimum Annealing Temperature melting temperature UV-spectrophotometer UV transilluminator World Health Organization

VI

r

List of Tables

Table Page 31 The components that were added into the 151lL mixture of RNA sample 21

random primers and nuclease free water

32 The PCR Master Mix that was prepared for 7 reactions to test for Ta of 23 RPS15 primer set

33 The PCR Master Mix that was prepared for 5 reactions to test for Ta of 23 GAPDH primer set

34 The generalized representation for the components of test reactions and 23 negative control reactions

35 The parameters of PCR reaction for RPS15 24

36 The parameters ofPCR reaction for GAPDH 24

37 The components of the PCR Master Mix 26

38 The components ofthe test reaction and positive as well as negative control 26 reactions that were added to PCR Master Mix

39 The PCR profile of the Optimized PCR for all the reactions 26

41 The absorbance readings concentrations of RNA and absorbance ratios 29 for all the RNA extracted from NP69 and HKl

42 Band intensities of all the genes based on pixel volumes quantified using 34 ImageQuant TL Software

43 Nonnalized RPS15 values 36

VII

List of Figures

Figure Page Circled indicates the location ofRPS15 gene in chromosome 19 ofHomo

21 sapiens (Adapted from httpwwwncbinlmnihgovgene6209) 8

RNA Integrity Checking through AGE (A) The gel image of intact fresh RNA extracted from HKl as viewed on UV-transilluminator (B) The gel

41 image of intact RNA extracted from NP69 captured by iQuant Capture 28 Software

Amplification of GAPDH using gradient PCR to detennine the T aOpt

42 (Lane 1=493degC Lane 2=507degC Lane 3=530oC) 30

Amplification ofRPS15 using gradient PCR to detennine the T aOpt (Lane 1=570oC Lane 2=576degC Lane 3=604degC Lane 4=642degC Lane43 305=660oC)

Analysis of expression of RPS15 in nonnal and tumour nasopharyngeal 44 epithelium cell lines with the positive control GAPDHusing RT-PCR 33

Graphical representation on the pixel volumes of each genes in each cell lines plotted together with the mean and error bars from the average of45 35the readings of each genes in each cell lines

Average of the nonnalized RPS15 values in NP69 nonnal cell line and HK1 tumour cell line NP69 plot n=2 HKl plot n=2 Two sample 46 36Unpaired I-test 2 degree of freedom 95 confident level plt005

VIII

I declare that ProjectThesis is classified as (Please tick (Jraquo

D CONFIDENTIAL (Contains confidential information under the Official Secret Act 1972) DRESTRICTED (Contains restricted information as specified by the organization where

research was done) ~OPEN ACCESS

Validation of ProjectThesis

I therefore duly affirm with free consent and willingly declare that this said ProjectlThesis shall be placed officially in the Centre for Academic Information Services with the abiding interest and rights as follows

bull This ProjectlThesis IS the sole legal property of Universiti Malaysia Sarawak (UNlMAS)

bull The Centre for Academic Information Services has the lawful right to make copies for the purpose of academic and research only and not for other purpose

bull The Centre for Academic Information Services has the lawful right to digitalize the content for the Local Content Database

bull The Centre for Academic Information Services has the lawful right to make copies of the ProjectThesis for academic exchange between Higher Learning Institute

bull No dispute or any claim shall arise from the student itself neither third party on this ProjectlThesis once it becomes the sole property of UNlMAS

bull This ProjectlThesis or any material data and information related to it shall not be distributed published or disclosed to any party by the student except with UNlMAS permISSIon

-

~ Student signature llL 2 21tltJl Supervisor signature L-a middot _ shy

(Date) (Date)

Current Address 6 TAMAN MA WAR JALAN MA WAR

06700 PENDANG KEDAH DARUL AMAN

Notes If the ProjectThesis is CONFIDENTIAL or RESTRICTED please attach together as annexure a letter from the organization with the period and reasons of confidentiality and restriction

[The instrument is duly prepared by The Centre for Academic Information Services]

III

Pusat Khidmat Maklumat Akad~mi~ UNlVERSm MALAYSIA SARAWAt~

Table of Content

Contents Page Title and Front Cover Acknowledgement I Declaration II Content Page IV List of Abbreviations VI List of Tables VII List of Figures VIII Abstract in English and Bahasa Malaysia 1 10 Introduction 2

11 Background 2 12 Objectives 3

20 Literature Review 4 21 Nasopharyngeal Carcinoma (NPC) 4

211 Causative Factors ofNPC 4 212 Staging ofNPC 5 213 Histopathological types ofNPC 5 214 Genes Linked to NPC 6

22 Ribosomal Proteins (RPs) 7 221 Extra-ribosomal functions ofRPs 7

23 Ribosomal Protein S 15 (RPS 15) 8 231 The Relevance ofRPS15 with Various Hematologic

Disorders 9 2311 Mutation ofRPs caused ribosomopathies 9 2312 Interaction ofRPs with p53 9

24 Cell Lines Used in RPS 15 Gene Expression Level Analysis 10 241 NP69 cell line 10 242 NPCHKl cell line I t

25 Analysis of Gene Expression 11 251 Housekeeping gene as a control 11

2511 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene 12

26 Principles of Methodology 12 261 The Advantages of TRIzol Reagent 12 262 Qualitative and Quantitative Analysis of RNA 13 263 Considerations on Designing Primer 14 264 Optimization of Primer Annealing Temperature 15 265 Direct Sequencing ofPCR Products 15 266 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 16

IV

30 Methodology 18 31 RNA Extraction 18 32 RNA Confinnation Steps 19

321 Quantifying the Concentration of RNA 19 322 Checking RNA Quality using Agarose Gel Electrophoresis 20

(AGE) 33 Reverse Transcription of RNA sample 20 34 Designing Primer Sets for GAPDH and RPSJ5 21 35 Gradient PCR for Optimization of Primer Annealing Temperature 22 36 Sequencing of Amplified PCR Products 24 37 Optimized RT-PCR for Gene Expression Analysis 25 38 Analysis of Band Intensities 27

40 Results 28 41 Qualitative and Quantitative Analysis of RNA Samples 28 42 Gradient PCR for Optimization on the Annealing Temperature of 29GAPDH and RPSJ5 43 The Identity of Amplified Sequences 31 44 Optimized RT-PCR for Analysis of Gene Expression Levels 32 45 Statistical Data Analysis 33

50 Discussion 38 51 The Possible Effects on Overexpression ofRP Gene 41 52 Limitations ofRT-PCR 42

60 Conclusion 44 61 Recommendations 44

70 References 45

Appendices 50 Appendix 1 Various Requirements for Primer Design 50 Appendix 2 Sequencing Results for RPSJ5 and GAPDH 54 Appendix 3 Band Intensities Analysis 64 Appendix 4 Statistical Data Analysis 65

V

1 1

List of Abbreviations

A260

A280

AGE Cdk eDNA eLL Co-IP DBA BBV EtBr GAPDH HCC HLA ICAT LMPI MALDI-TOF Mdm2 M-MLVRT NOMA NP NPC NPIP NPYR NTC PCR PTEN RING RNA RP RPS15 rRNA RT-PCR SCC Spl Ta TAE Taopi Tm UV-spec UV-trans WHO

Absorbance at 260nm Absorbance at 280nm Agarose Ge1 Electrophoresis CyeliD-dependent kinase complementary DNA Chronic lymphocytic leukemia Co-Immunoprecipitation Dark-Blackfan Anaemia Epstein-Barr Virus Ethidium Bromide Glyceraldehyde-3-Phosphate Dehydrogenase Human Hepatocellular Carcinoma Human Leukocyte Antigen Isotope-coded Affinity Tag EBV -encoded latent membrane protein 1 Matrix-Assisted Laser Desorption Ionization with Time-of-Flight Mouse double minute 2 Moloney Murine Leukemia Virus Reverse Transcriptase N i trosodimethyamine Nasopharyngeal Nasopharyngeal Carcinoma N -nitrospiperidine N -nitrospyrrolidene Non-template control Polymerase Chain Reaction Phosphatase and tensin homolog Really Interesting New Gene Ribonueleic Acid Ribosomal Protein Ribosomal Protein S 15 ribosomal RNA Reverse Transcription Polymerase Chain Reaction Squamous Cell Carcinoma Specific protein 1 Annealing Temperature Tris-Acetate-Ethylenediamine-Tetraacetic-Acid Optimum Annealing Temperature melting temperature UV-spectrophotometer UV transilluminator World Health Organization

VI

r

List of Tables

Table Page 31 The components that were added into the 151lL mixture of RNA sample 21

random primers and nuclease free water

32 The PCR Master Mix that was prepared for 7 reactions to test for Ta of 23 RPS15 primer set

33 The PCR Master Mix that was prepared for 5 reactions to test for Ta of 23 GAPDH primer set

34 The generalized representation for the components of test reactions and 23 negative control reactions

35 The parameters of PCR reaction for RPS15 24

36 The parameters ofPCR reaction for GAPDH 24

37 The components of the PCR Master Mix 26

38 The components ofthe test reaction and positive as well as negative control 26 reactions that were added to PCR Master Mix

39 The PCR profile of the Optimized PCR for all the reactions 26

41 The absorbance readings concentrations of RNA and absorbance ratios 29 for all the RNA extracted from NP69 and HKl

42 Band intensities of all the genes based on pixel volumes quantified using 34 ImageQuant TL Software

43 Nonnalized RPS15 values 36

VII

List of Figures

Figure Page Circled indicates the location ofRPS15 gene in chromosome 19 ofHomo

21 sapiens (Adapted from httpwwwncbinlmnihgovgene6209) 8

RNA Integrity Checking through AGE (A) The gel image of intact fresh RNA extracted from HKl as viewed on UV-transilluminator (B) The gel

41 image of intact RNA extracted from NP69 captured by iQuant Capture 28 Software

Amplification of GAPDH using gradient PCR to detennine the T aOpt

42 (Lane 1=493degC Lane 2=507degC Lane 3=530oC) 30

Amplification ofRPS15 using gradient PCR to detennine the T aOpt (Lane 1=570oC Lane 2=576degC Lane 3=604degC Lane 4=642degC Lane43 305=660oC)

Analysis of expression of RPS15 in nonnal and tumour nasopharyngeal 44 epithelium cell lines with the positive control GAPDHusing RT-PCR 33

Graphical representation on the pixel volumes of each genes in each cell lines plotted together with the mean and error bars from the average of45 35the readings of each genes in each cell lines

Average of the nonnalized RPS15 values in NP69 nonnal cell line and HK1 tumour cell line NP69 plot n=2 HKl plot n=2 Two sample 46 36Unpaired I-test 2 degree of freedom 95 confident level plt005

VIII

Pusat Khidmat Maklumat Akad~mi~ UNlVERSm MALAYSIA SARAWAt~

Table of Content

Contents Page Title and Front Cover Acknowledgement I Declaration II Content Page IV List of Abbreviations VI List of Tables VII List of Figures VIII Abstract in English and Bahasa Malaysia 1 10 Introduction 2

11 Background 2 12 Objectives 3

20 Literature Review 4 21 Nasopharyngeal Carcinoma (NPC) 4

211 Causative Factors ofNPC 4 212 Staging ofNPC 5 213 Histopathological types ofNPC 5 214 Genes Linked to NPC 6

22 Ribosomal Proteins (RPs) 7 221 Extra-ribosomal functions ofRPs 7

23 Ribosomal Protein S 15 (RPS 15) 8 231 The Relevance ofRPS15 with Various Hematologic

Disorders 9 2311 Mutation ofRPs caused ribosomopathies 9 2312 Interaction ofRPs with p53 9

24 Cell Lines Used in RPS 15 Gene Expression Level Analysis 10 241 NP69 cell line 10 242 NPCHKl cell line I t

25 Analysis of Gene Expression 11 251 Housekeeping gene as a control 11

2511 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene 12

26 Principles of Methodology 12 261 The Advantages of TRIzol Reagent 12 262 Qualitative and Quantitative Analysis of RNA 13 263 Considerations on Designing Primer 14 264 Optimization of Primer Annealing Temperature 15 265 Direct Sequencing ofPCR Products 15 266 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 16

IV

30 Methodology 18 31 RNA Extraction 18 32 RNA Confinnation Steps 19

321 Quantifying the Concentration of RNA 19 322 Checking RNA Quality using Agarose Gel Electrophoresis 20

(AGE) 33 Reverse Transcription of RNA sample 20 34 Designing Primer Sets for GAPDH and RPSJ5 21 35 Gradient PCR for Optimization of Primer Annealing Temperature 22 36 Sequencing of Amplified PCR Products 24 37 Optimized RT-PCR for Gene Expression Analysis 25 38 Analysis of Band Intensities 27

40 Results 28 41 Qualitative and Quantitative Analysis of RNA Samples 28 42 Gradient PCR for Optimization on the Annealing Temperature of 29GAPDH and RPSJ5 43 The Identity of Amplified Sequences 31 44 Optimized RT-PCR for Analysis of Gene Expression Levels 32 45 Statistical Data Analysis 33

50 Discussion 38 51 The Possible Effects on Overexpression ofRP Gene 41 52 Limitations ofRT-PCR 42

60 Conclusion 44 61 Recommendations 44

70 References 45

Appendices 50 Appendix 1 Various Requirements for Primer Design 50 Appendix 2 Sequencing Results for RPSJ5 and GAPDH 54 Appendix 3 Band Intensities Analysis 64 Appendix 4 Statistical Data Analysis 65

V

1 1

List of Abbreviations

A260

A280

AGE Cdk eDNA eLL Co-IP DBA BBV EtBr GAPDH HCC HLA ICAT LMPI MALDI-TOF Mdm2 M-MLVRT NOMA NP NPC NPIP NPYR NTC PCR PTEN RING RNA RP RPS15 rRNA RT-PCR SCC Spl Ta TAE Taopi Tm UV-spec UV-trans WHO

Absorbance at 260nm Absorbance at 280nm Agarose Ge1 Electrophoresis CyeliD-dependent kinase complementary DNA Chronic lymphocytic leukemia Co-Immunoprecipitation Dark-Blackfan Anaemia Epstein-Barr Virus Ethidium Bromide Glyceraldehyde-3-Phosphate Dehydrogenase Human Hepatocellular Carcinoma Human Leukocyte Antigen Isotope-coded Affinity Tag EBV -encoded latent membrane protein 1 Matrix-Assisted Laser Desorption Ionization with Time-of-Flight Mouse double minute 2 Moloney Murine Leukemia Virus Reverse Transcriptase N i trosodimethyamine Nasopharyngeal Nasopharyngeal Carcinoma N -nitrospiperidine N -nitrospyrrolidene Non-template control Polymerase Chain Reaction Phosphatase and tensin homolog Really Interesting New Gene Ribonueleic Acid Ribosomal Protein Ribosomal Protein S 15 ribosomal RNA Reverse Transcription Polymerase Chain Reaction Squamous Cell Carcinoma Specific protein 1 Annealing Temperature Tris-Acetate-Ethylenediamine-Tetraacetic-Acid Optimum Annealing Temperature melting temperature UV-spectrophotometer UV transilluminator World Health Organization

VI

r

List of Tables

Table Page 31 The components that were added into the 151lL mixture of RNA sample 21

random primers and nuclease free water

32 The PCR Master Mix that was prepared for 7 reactions to test for Ta of 23 RPS15 primer set

33 The PCR Master Mix that was prepared for 5 reactions to test for Ta of 23 GAPDH primer set

34 The generalized representation for the components of test reactions and 23 negative control reactions

35 The parameters of PCR reaction for RPS15 24

36 The parameters ofPCR reaction for GAPDH 24

37 The components of the PCR Master Mix 26

38 The components ofthe test reaction and positive as well as negative control 26 reactions that were added to PCR Master Mix

39 The PCR profile of the Optimized PCR for all the reactions 26

41 The absorbance readings concentrations of RNA and absorbance ratios 29 for all the RNA extracted from NP69 and HKl

42 Band intensities of all the genes based on pixel volumes quantified using 34 ImageQuant TL Software

43 Nonnalized RPS15 values 36

VII

List of Figures

Figure Page Circled indicates the location ofRPS15 gene in chromosome 19 ofHomo

21 sapiens (Adapted from httpwwwncbinlmnihgovgene6209) 8

RNA Integrity Checking through AGE (A) The gel image of intact fresh RNA extracted from HKl as viewed on UV-transilluminator (B) The gel

41 image of intact RNA extracted from NP69 captured by iQuant Capture 28 Software

Amplification of GAPDH using gradient PCR to detennine the T aOpt

42 (Lane 1=493degC Lane 2=507degC Lane 3=530oC) 30

Amplification ofRPS15 using gradient PCR to detennine the T aOpt (Lane 1=570oC Lane 2=576degC Lane 3=604degC Lane 4=642degC Lane43 305=660oC)

Analysis of expression of RPS15 in nonnal and tumour nasopharyngeal 44 epithelium cell lines with the positive control GAPDHusing RT-PCR 33

Graphical representation on the pixel volumes of each genes in each cell lines plotted together with the mean and error bars from the average of45 35the readings of each genes in each cell lines

Average of the nonnalized RPS15 values in NP69 nonnal cell line and HK1 tumour cell line NP69 plot n=2 HKl plot n=2 Two sample 46 36Unpaired I-test 2 degree of freedom 95 confident level plt005

VIII

30 Methodology 18 31 RNA Extraction 18 32 RNA Confinnation Steps 19

321 Quantifying the Concentration of RNA 19 322 Checking RNA Quality using Agarose Gel Electrophoresis 20

(AGE) 33 Reverse Transcription of RNA sample 20 34 Designing Primer Sets for GAPDH and RPSJ5 21 35 Gradient PCR for Optimization of Primer Annealing Temperature 22 36 Sequencing of Amplified PCR Products 24 37 Optimized RT-PCR for Gene Expression Analysis 25 38 Analysis of Band Intensities 27

40 Results 28 41 Qualitative and Quantitative Analysis of RNA Samples 28 42 Gradient PCR for Optimization on the Annealing Temperature of 29GAPDH and RPSJ5 43 The Identity of Amplified Sequences 31 44 Optimized RT-PCR for Analysis of Gene Expression Levels 32 45 Statistical Data Analysis 33

50 Discussion 38 51 The Possible Effects on Overexpression ofRP Gene 41 52 Limitations ofRT-PCR 42

60 Conclusion 44 61 Recommendations 44

70 References 45

Appendices 50 Appendix 1 Various Requirements for Primer Design 50 Appendix 2 Sequencing Results for RPSJ5 and GAPDH 54 Appendix 3 Band Intensities Analysis 64 Appendix 4 Statistical Data Analysis 65

V

1 1

List of Abbreviations

A260

A280

AGE Cdk eDNA eLL Co-IP DBA BBV EtBr GAPDH HCC HLA ICAT LMPI MALDI-TOF Mdm2 M-MLVRT NOMA NP NPC NPIP NPYR NTC PCR PTEN RING RNA RP RPS15 rRNA RT-PCR SCC Spl Ta TAE Taopi Tm UV-spec UV-trans WHO

Absorbance at 260nm Absorbance at 280nm Agarose Ge1 Electrophoresis CyeliD-dependent kinase complementary DNA Chronic lymphocytic leukemia Co-Immunoprecipitation Dark-Blackfan Anaemia Epstein-Barr Virus Ethidium Bromide Glyceraldehyde-3-Phosphate Dehydrogenase Human Hepatocellular Carcinoma Human Leukocyte Antigen Isotope-coded Affinity Tag EBV -encoded latent membrane protein 1 Matrix-Assisted Laser Desorption Ionization with Time-of-Flight Mouse double minute 2 Moloney Murine Leukemia Virus Reverse Transcriptase N i trosodimethyamine Nasopharyngeal Nasopharyngeal Carcinoma N -nitrospiperidine N -nitrospyrrolidene Non-template control Polymerase Chain Reaction Phosphatase and tensin homolog Really Interesting New Gene Ribonueleic Acid Ribosomal Protein Ribosomal Protein S 15 ribosomal RNA Reverse Transcription Polymerase Chain Reaction Squamous Cell Carcinoma Specific protein 1 Annealing Temperature Tris-Acetate-Ethylenediamine-Tetraacetic-Acid Optimum Annealing Temperature melting temperature UV-spectrophotometer UV transilluminator World Health Organization

VI

r

List of Tables

Table Page 31 The components that were added into the 151lL mixture of RNA sample 21

random primers and nuclease free water

32 The PCR Master Mix that was prepared for 7 reactions to test for Ta of 23 RPS15 primer set

33 The PCR Master Mix that was prepared for 5 reactions to test for Ta of 23 GAPDH primer set

34 The generalized representation for the components of test reactions and 23 negative control reactions

35 The parameters of PCR reaction for RPS15 24

36 The parameters ofPCR reaction for GAPDH 24

37 The components of the PCR Master Mix 26

38 The components ofthe test reaction and positive as well as negative control 26 reactions that were added to PCR Master Mix

39 The PCR profile of the Optimized PCR for all the reactions 26

41 The absorbance readings concentrations of RNA and absorbance ratios 29 for all the RNA extracted from NP69 and HKl

42 Band intensities of all the genes based on pixel volumes quantified using 34 ImageQuant TL Software

43 Nonnalized RPS15 values 36

VII

List of Figures

Figure Page Circled indicates the location ofRPS15 gene in chromosome 19 ofHomo

21 sapiens (Adapted from httpwwwncbinlmnihgovgene6209) 8

RNA Integrity Checking through AGE (A) The gel image of intact fresh RNA extracted from HKl as viewed on UV-transilluminator (B) The gel

41 image of intact RNA extracted from NP69 captured by iQuant Capture 28 Software

Amplification of GAPDH using gradient PCR to detennine the T aOpt

42 (Lane 1=493degC Lane 2=507degC Lane 3=530oC) 30

Amplification ofRPS15 using gradient PCR to detennine the T aOpt (Lane 1=570oC Lane 2=576degC Lane 3=604degC Lane 4=642degC Lane43 305=660oC)

Analysis of expression of RPS15 in nonnal and tumour nasopharyngeal 44 epithelium cell lines with the positive control GAPDHusing RT-PCR 33

Graphical representation on the pixel volumes of each genes in each cell lines plotted together with the mean and error bars from the average of45 35the readings of each genes in each cell lines

Average of the nonnalized RPS15 values in NP69 nonnal cell line and HK1 tumour cell line NP69 plot n=2 HKl plot n=2 Two sample 46 36Unpaired I-test 2 degree of freedom 95 confident level plt005

VIII

1 1

List of Abbreviations

A260

A280

AGE Cdk eDNA eLL Co-IP DBA BBV EtBr GAPDH HCC HLA ICAT LMPI MALDI-TOF Mdm2 M-MLVRT NOMA NP NPC NPIP NPYR NTC PCR PTEN RING RNA RP RPS15 rRNA RT-PCR SCC Spl Ta TAE Taopi Tm UV-spec UV-trans WHO

Absorbance at 260nm Absorbance at 280nm Agarose Ge1 Electrophoresis CyeliD-dependent kinase complementary DNA Chronic lymphocytic leukemia Co-Immunoprecipitation Dark-Blackfan Anaemia Epstein-Barr Virus Ethidium Bromide Glyceraldehyde-3-Phosphate Dehydrogenase Human Hepatocellular Carcinoma Human Leukocyte Antigen Isotope-coded Affinity Tag EBV -encoded latent membrane protein 1 Matrix-Assisted Laser Desorption Ionization with Time-of-Flight Mouse double minute 2 Moloney Murine Leukemia Virus Reverse Transcriptase N i trosodimethyamine Nasopharyngeal Nasopharyngeal Carcinoma N -nitrospiperidine N -nitrospyrrolidene Non-template control Polymerase Chain Reaction Phosphatase and tensin homolog Really Interesting New Gene Ribonueleic Acid Ribosomal Protein Ribosomal Protein S 15 ribosomal RNA Reverse Transcription Polymerase Chain Reaction Squamous Cell Carcinoma Specific protein 1 Annealing Temperature Tris-Acetate-Ethylenediamine-Tetraacetic-Acid Optimum Annealing Temperature melting temperature UV-spectrophotometer UV transilluminator World Health Organization

VI

r

List of Tables

Table Page 31 The components that were added into the 151lL mixture of RNA sample 21

random primers and nuclease free water

32 The PCR Master Mix that was prepared for 7 reactions to test for Ta of 23 RPS15 primer set

33 The PCR Master Mix that was prepared for 5 reactions to test for Ta of 23 GAPDH primer set

34 The generalized representation for the components of test reactions and 23 negative control reactions

35 The parameters of PCR reaction for RPS15 24

36 The parameters ofPCR reaction for GAPDH 24

37 The components of the PCR Master Mix 26

38 The components ofthe test reaction and positive as well as negative control 26 reactions that were added to PCR Master Mix

39 The PCR profile of the Optimized PCR for all the reactions 26

41 The absorbance readings concentrations of RNA and absorbance ratios 29 for all the RNA extracted from NP69 and HKl

42 Band intensities of all the genes based on pixel volumes quantified using 34 ImageQuant TL Software

43 Nonnalized RPS15 values 36

VII

List of Figures

Figure Page Circled indicates the location ofRPS15 gene in chromosome 19 ofHomo

21 sapiens (Adapted from httpwwwncbinlmnihgovgene6209) 8

RNA Integrity Checking through AGE (A) The gel image of intact fresh RNA extracted from HKl as viewed on UV-transilluminator (B) The gel

41 image of intact RNA extracted from NP69 captured by iQuant Capture 28 Software

Amplification of GAPDH using gradient PCR to detennine the T aOpt

42 (Lane 1=493degC Lane 2=507degC Lane 3=530oC) 30

Amplification ofRPS15 using gradient PCR to detennine the T aOpt (Lane 1=570oC Lane 2=576degC Lane 3=604degC Lane 4=642degC Lane43 305=660oC)

Analysis of expression of RPS15 in nonnal and tumour nasopharyngeal 44 epithelium cell lines with the positive control GAPDHusing RT-PCR 33

Graphical representation on the pixel volumes of each genes in each cell lines plotted together with the mean and error bars from the average of45 35the readings of each genes in each cell lines

Average of the nonnalized RPS15 values in NP69 nonnal cell line and HK1 tumour cell line NP69 plot n=2 HKl plot n=2 Two sample 46 36Unpaired I-test 2 degree of freedom 95 confident level plt005

VIII

r

List of Tables

Table Page 31 The components that were added into the 151lL mixture of RNA sample 21

random primers and nuclease free water

32 The PCR Master Mix that was prepared for 7 reactions to test for Ta of 23 RPS15 primer set

33 The PCR Master Mix that was prepared for 5 reactions to test for Ta of 23 GAPDH primer set

34 The generalized representation for the components of test reactions and 23 negative control reactions

35 The parameters of PCR reaction for RPS15 24

36 The parameters ofPCR reaction for GAPDH 24

37 The components of the PCR Master Mix 26

38 The components ofthe test reaction and positive as well as negative control 26 reactions that were added to PCR Master Mix

39 The PCR profile of the Optimized PCR for all the reactions 26

41 The absorbance readings concentrations of RNA and absorbance ratios 29 for all the RNA extracted from NP69 and HKl

42 Band intensities of all the genes based on pixel volumes quantified using 34 ImageQuant TL Software

43 Nonnalized RPS15 values 36

VII

List of Figures

Figure Page Circled indicates the location ofRPS15 gene in chromosome 19 ofHomo

21 sapiens (Adapted from httpwwwncbinlmnihgovgene6209) 8

RNA Integrity Checking through AGE (A) The gel image of intact fresh RNA extracted from HKl as viewed on UV-transilluminator (B) The gel

41 image of intact RNA extracted from NP69 captured by iQuant Capture 28 Software

Amplification of GAPDH using gradient PCR to detennine the T aOpt

42 (Lane 1=493degC Lane 2=507degC Lane 3=530oC) 30

Amplification ofRPS15 using gradient PCR to detennine the T aOpt (Lane 1=570oC Lane 2=576degC Lane 3=604degC Lane 4=642degC Lane43 305=660oC)

Analysis of expression of RPS15 in nonnal and tumour nasopharyngeal 44 epithelium cell lines with the positive control GAPDHusing RT-PCR 33

Graphical representation on the pixel volumes of each genes in each cell lines plotted together with the mean and error bars from the average of45 35the readings of each genes in each cell lines

Average of the nonnalized RPS15 values in NP69 nonnal cell line and HK1 tumour cell line NP69 plot n=2 HKl plot n=2 Two sample 46 36Unpaired I-test 2 degree of freedom 95 confident level plt005

VIII

List of Figures

Figure Page Circled indicates the location ofRPS15 gene in chromosome 19 ofHomo

21 sapiens (Adapted from httpwwwncbinlmnihgovgene6209) 8

RNA Integrity Checking through AGE (A) The gel image of intact fresh RNA extracted from HKl as viewed on UV-transilluminator (B) The gel

41 image of intact RNA extracted from NP69 captured by iQuant Capture 28 Software

Amplification of GAPDH using gradient PCR to detennine the T aOpt

42 (Lane 1=493degC Lane 2=507degC Lane 3=530oC) 30

Amplification ofRPS15 using gradient PCR to detennine the T aOpt (Lane 1=570oC Lane 2=576degC Lane 3=604degC Lane 4=642degC Lane43 305=660oC)

Analysis of expression of RPS15 in nonnal and tumour nasopharyngeal 44 epithelium cell lines with the positive control GAPDHusing RT-PCR 33

Graphical representation on the pixel volumes of each genes in each cell lines plotted together with the mean and error bars from the average of45 35the readings of each genes in each cell lines

Average of the nonnalized RPS15 values in NP69 nonnal cell line and HK1 tumour cell line NP69 plot n=2 HKl plot n=2 Two sample 46 36Unpaired I-test 2 degree of freedom 95 confident level plt005

VIII

Comparison of RPS15 Expression Levels between Normal and Tumour CeUs of Human Nasopharyngeal Epithelium

ONG LIAN WEEN

Resource Biotechnology Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARA W AIlt

ABSTRACT

Ribosomal protein S 15 (RPS15) gene is known as protein-coding gene RPS15 encodes for the ribosomal protein which is a constituent of small ribosomal subunit 40S of ribosome Recent studies have shown that RPS15 interact with Mdm2 which inhibits the degradation ofp53 a tumour suppressor Furthermore mutation in RPS15 gene have been reported to be involved in Dark-Blackfan Anaemia (DBA) This is evidence ofRPS15 gene involvement in clinical diseases The clinical cases that drawn our interest in this study is nasopharyngeal carcinoma (NPC) A study has mentioned that various RPs are involved in NPC But the information about association of RPS15 in NPC is still limited Therefore in this study we examine the expression level of RPS15 gene in normal and tumour nasopharyngeal epithelium The cell lines that were used in this research are NP69 and HKI which are normal and tumour cell lines of nasopharyngeal respectively In this study the expression levels of RPS15 gene in both cell lines were analyzed using reverse transcriptase polymerase chain reaction (RT-PCR) RPS15 gene was found to be overexpressed in HKI compared to NP69 as the fold difference recorded to be greater than I Moreover statistical analysis using independent Student t test showed that the differential expression of RPS15 is significant The overexpression of RPS15 gene in nasopharyngeal tumour cell line suggested the involvement ofRPS15 in the development ofNPC as it was found to have biological function in causing cancer in recent study The study ofgene expression level ofRPS15 is expected to contribute in the effort to identify cancer biomarkers in the future

Keywords RPS15 NPC gene expression level housekeeping gene RT-PCR

ABSTRAK

Gen Protein Ribosom S15 (RPS15) dikenali sebagai gen yang mengekod protein RPS15 mengekod protein ribosom yang merupakan satu daripada komponen subunit kecil 40S daripada ribosom Kajian terbaru menunjukkan bahawa RPS15 berinteraksi dengan Mdm2 yang menghalang degradasi p53 penindas tumor Tambahan pula mutasi dalam gen RPS15 telah dilaporkan terlibat dalam Anemia DarkshyBlackfan (DBA) Hal-hal tersebut membuktikan penglibatan gen RPS15 dalam penyakit klinikal Kes Iclillikal yang menarik minat kami dalam kajian ini adalah karsinoma nasofarinks (NPC) Satu kajian lelah mendapati bahawa pelbagai RP terlibat dalam NPC Tetapi maklumat mengenai hubungkait RPS1 5 dengan NPC masih terhad Oleh itu dalam kajian ini kami mengkaji tahap ungkapan gen RPS15 daam epitelium nasofarinks normal dan tumor Bahagian-bahagian sel yang digunakan dalam kajian ini adalah NP69 dan HK1 yang mengwakili sel nasofarinks normal dan tumor masing-masing Dalam kajian ini tahap ekspresi gen RPS15 dalam kedua-dua bahagian sel dianalisis menggunakan tindak balas rantai polimeras transkriptasi terbalik (RT-PCR) mendapati bahawa RPS15 lebih terungkap dalam HK1 berbanding NP69 kerana perbezaan kali ganda dicatatkan lebih besar daripada 1 Selain itu analisis stalistik menggunakan bebas ujian t Pelajar menunjukkan bahawa perbezaan ungkapan RPS15 adalah penting Justeru mencadangkan penglibatan RPS15 dalam pembentukan NPC kerana ia didapali mempunyaifungsi biologi yang menyebabkan kanser dalam kajian baru-baru ini Kajian mengenai tahap gen ungkapan RPS15 dijangka menyumbang dalam usaha untuk mengenal pasti penanda kanser pada masa hadapan

Kala kunci RPS15 NPC tahap ungkapan gen gen pengemasan RT-PCR

1

10 Introduction

11 Background

Nasopharyngeal carcinoma (NPC) is a cancer that commonly found to affect the upper part

ofpharynx It is found to have high occurrence in children and adults The cases ofNPC was

ranked the third common cancer that affected male population in Malaysia (Omar amp Tamin

201 1)

Traditionally ribosomal proteins (RPs) functions in constituting stoichiometric

amounts of ribosome together with ribosomal RNA (rRNA) which involved in cellular

translation This stoichiometric amounts of ribosome is recognized as ribosomal subunits

Apart from the role ofRPs in ribosome biogenesis RPs was also found to have extrashy

ribosomal functions which include DNA replication repair and transcription modifications

ofRNA proliferation and cell growth apoptosis regulation and cellular transformation (Lai

amp Xu 2007)

Ribosomal protein S 15 (RPS 15) is a protein coding gene It produces ribosomal

protein constituted in small 40S subunit of ribosome (Robledo et at 2008) RPS 15 is found

to be involved in a congenital erythroid aplasia which is also known as Diamond-Blankfan

anemia (DBA) (Bhavsar Makley amp Tsonis 2010) In addition study on human

hepatocellular carcinoma (HCC) found that there was a relation between RPS15 gene

expressions with this disease (Yoon et at 2006)

Recent studies stated that cancer developed in human is related to ribosomal proteins

(RPs) (Daftuar Zhu Jacq amp Prives 2013) The study using zebrafish (Danio rerio) as an

animal model has shown that several RP genes are cancer genes in this organism

2

(Amsterdam et al 2004) A recent study has shown that expression level of various RPs

was either up-regulated or down-regulated in colorectal carcinoma (CRC) (Lai amp Xu 2007)

There were several RP genes that were found to be downregulated in NPC namely

RPS26 and RPS27 (Sim Toh amp Tiong 2008) Currently there is a lack of findings on the

relationship between the gene expression of RPS15 and nasopharyngeal cancer

This research is aimed to compare the differential expression level of RPS15 gene in

normal NP69 cell line and tumour HKI cell line The RPS15 is expected to show

significant difference in gene expression in comparison between normal and NPC cell line

The study on gene expression of RPs is in the hope to bring a breakthrough in the

study of cancer Hopefully gene expression of RPs will contributes to future cancer

researches such as in biomarker assessment gene manipulation and silencing in cancer

therapy

12 Objectives

The objectives throughout the completion of this project are of the following

1 To detect the expression of RPS15 gene in NP69 normal nasopharyngeal cell line and

HKI NPC cell line

2 To compare the differential expression level of RPS15 gene in NP69 normal human

nasopharyngeal cell line and HKI NPC cell line

3

l I

20 Literature Review

21 Nasopharyngeal Carcinoma (NPC)

Zeng and Zeng (2010) mentioned that NPC is found at the Eustachian tube surrounding

ostium that is situated in the nasopharynx lateral wall This disease have a bimodal

distribution in the age group where it affects people between age of 50 to 60 and children at

late childhood NPC also found higher in men than women (Zeng amp Zeng 2010)

211 Causative Factors of NPC

The major causes of NPC are geographical and population factor environmental factor

genetic factors and meal consumption factor (Zeng amp Zeng 2010) NPC was recorded to

affect population regionally and geographically as there was a highest rates of occurrence in

Southern Chinese population in Guangdong Greelanders native and Inuits of Alaska

compared to lower occurrence among population in Caucasians from Western countries

(Zeng amp Zeng 2010) Besides genetic factor was found to be causative factor due to familial

NPC history among Cantonese population (Zeng amp Zeng 2010) The haplotypes which is

the inherited genes from a single parent ofhuman leukocyte antigen (HLA) detected among

Chinese population who were being diagnosed with NPC (Zeng amp Zeng 2010) Moreover

components inside preserved food for example N-nitrospiperidine (NPIP)

nitrosodimethyamine (NDMA) and N-nitrospyrrolidene (NPYR) are the causative agents in

developing NPC (Zeng amp Zeng 2010) Another causative factor of NPC was caused by

DNA tumour virus which was the Epstein - Barr virus (EBV) infection This is because viral

genome ofEBV was found in the carcinoma cells ofNPC (Feinmesser et ai 1992)

4

Pusat Khidmat Maklumat Akad(~v UNlVEltSm MALAYSIA SAR4WAK

212 Staging of NPC

According to Hu (2010) NPC spread easily through lymph system resulted in early lymph

node around the neck Thus NPC is highly metastatic The chance of recovery depends

greatly on the staging of the cancer whether cancer has spread through the enlargement of

tumour size involvement of lymph node and metastasis (Hu 2010) Stage I NPC is a small

tumour found regionally in the nasopharynx Stage II NPC is a tumour found to extend from

nasopharynx to the oropharynx or spread to one or more lymph nodes on the side of the neck

Stage III NPC is a large tumour that has spread to surrounding sinuses or spread to lymph

nodes on both sides ofthe neck Stage IV NPC is large tumour that spread out ofnasopharynx

region or affected lymph nodes becomes larger with distant metastasis Hu (2010) mentioned

that patients from early stage have better prognosis than patient in stage III or higher due to

distant metastasis and lymph node involvement

213 Histopathological types of NPC

Three histological subtypes of NPC have been categorized by World Health Organization

(WHO) according to differentiation of cells (Zeng amp Zeng 2010)

First Type 1 under electron microscope there is intracellular bridges found in

squamous cell carcinoma (SCC) and good cell differentiation that makes keratin (Zeng amp

Zeng 2010) Type 2 is a squamous carcinoma that does not makes keratin and have cell

differentiation varied from mature to anaplastic (Zeng amp Zeng 2010) While Type 3 the

cells types are inconsistent and variable This undifferentiated and non-keratinizing Type 3

NPC found to make up the nasopharyngeal tumours (Zeng amp Zeng 2010) Zeng and Zeng

(2010) also stated that this tumour was considered as lymphoepithelioma due to its tumour

infiltration with lymphocytes Zeng and Zeng (2010) stated that type 3 NPC are more

common in Southern China while type 1 usually found in Western countries the nonshy

5

endemic areas In Type 3 NPC it was diagnosed with the presence ofEBV transcript and the

diagnosis was based on the location of tumour in nasopharynx (Zeng amp Zeng 2010)

However there was also another classification system suggested to group NPC based

on the state ofdifferentiation of NPC Flint et al (2010) suggested that NPC was able to be

classified into different type based on the state of differentiation namely Type 1 Type 2a

and Type 2b Well differentiated SCC that was keratinizing was grouped into Type 1 There

was slightly different between Type 2a and Type 2b Flint et al (2010) mentioned that SCC

that was non-keratinizing was classified as Type 2a While the non-keratinizing carcinoma

which was in the state of undifferentiated was classified as Type 2b

214 Genes Linked to NPC

There are a few RPs gene found to be associated with NPC According to Sim et al (2008)

the RPs gene that linked to NPC wereRPS26 and RPS27 The RPS26 was downregulated in

NPC cell which was more noticeable in Type II b NPC RPS27 also found to be

downregulated in Type II a and Type II b of NPC cell types in contrast to normal

nasopharyngeal cell (Sim et al 2008)

6

22 Ribosomal Proteins (RPs)

According to Robledo et a (2008) the combination of rRNA with RPs together with

nonribosomal protein and other factors are involved in the production of ribosomes Robledo

et al (2008) stated that subunits ofmature ribosome form through the association of the RPs

with the ribosomal genes that undergo transcription in nucleolus by RNA polymerase I The

subunits produced are 40S and 60S subunit (Robledo et al 2008)

Besides the contribution ofRPs in the making up of the subunits oforganelle in cell

it was found to have the extra-ribosomal functions (Bhavsar et a 20 I 0) The extrashy

ribosomal functions are for example replication splicing transcription and regulation of

life span This special cellular functions is independent of ribosomes (Bhavsar et al 2010)

Bhavsar et al (2010) mentioned that regulation of RPs are so special that it was able to

regulate its expression level and control cell cycle and apoptosis

221 Extra-ribosomal functions of RPs

Bhavsar et a (2010) pointed out that at different levels of gene regulation RPs able to

regulate its own expression as well as of other gene RPs took part in mechanisms of gene

regulation at different levels for example modification of chromatin transcription

translation post-translation modification and RNA processing (Bhavsar et a 2010)

Besides RPs also involved in replication of viral genome either DNA or RNA

viruses during viral infection (Bhavsar et al 2010) Bhavsar et al (2010) mentioned that

RPs also involved in DNA repair mechanism when DNA is damaged by cell metabolic

processes or environmental factors in both prokaryotes and eukaryotes

Moreover RPs are involved in the cell cycle regulation by acting together with the

regulatory molecules like cyclin-dependent kinase (Cdk) According to Bhavsar et a (2010)

7

Cdk controlled the advancement of cell to different phases of cell cycle The expression

levels of RPs also regulate cell cycle by activating apoptosis (Bhavsar et al 2010)

According to Boon and Sim (2015) apoptosis is known as normal process of cell death

Bhavsar et al (2010) stated that during apoptosis RPs interact with an ubiquitin ligase

Mouse double minute 2 (Mdm2) to validate a normal p53 level

23 Ribosomal Protein S15 (RPS15)

Kenmochi Ashworth Lennon Hisa and Tanaka (1998) found that several human RPs genes

are situated in chromosome RPS 15 gene is situated in chromosome 19 in human According

to Kenmochi et al (1998) the mapped position ofRPS15 gene in chromosome 19 is 19p 133

the distance from the top of short arm is 16 Mb and distance from the most distal short-arm

marker is 6cR

ll39726~ ~ l1490888~

~--------------~~--~----------------------~ GAKI tpSlS J C190rf25

DAZAP1------~I PCSK4 -shy~(C2 --------shy

Figure 21 Circled indicates the location of RPSI5 gene in chromosome 19 of Homo sapiens (Adapted from hupwwwncbinlmnihgovgene6209)

According to Bhavsar et al (2010) RPS 15 in Ecoli found to have extra-ribosomal

properties in which it is self-translation regulated The expression of mutated genes or

unusual level of expression of ribosomal proteins are related with disease disorder RPS 15

is found to be involved in a congenital erythroid aplasia which is also known as Diamond-

Blackfan anaemia (DBA) (Bhavsar et al 2010)

8

l

10 Introduction

11 Background

Nasopharyngeal carcinoma (NPC) is a cancer that commonly found to affect the upper part

ofpharynx It is found to have high occurrence in children and adults The cases ofNPC was

ranked the third common cancer that affected male population in Malaysia (Omar amp Tamin

201 1)

Traditionally ribosomal proteins (RPs) functions in constituting stoichiometric

amounts of ribosome together with ribosomal RNA (rRNA) which involved in cellular

translation This stoichiometric amounts of ribosome is recognized as ribosomal subunits

Apart from the role ofRPs in ribosome biogenesis RPs was also found to have extrashy

ribosomal functions which include DNA replication repair and transcription modifications

ofRNA proliferation and cell growth apoptosis regulation and cellular transformation (Lai

amp Xu 2007)

Ribosomal protein S 15 (RPS 15) is a protein coding gene It produces ribosomal

protein constituted in small 40S subunit of ribosome (Robledo et at 2008) RPS 15 is found

to be involved in a congenital erythroid aplasia which is also known as Diamond-Blankfan

anemia (DBA) (Bhavsar Makley amp Tsonis 2010) In addition study on human

hepatocellular carcinoma (HCC) found that there was a relation between RPS15 gene

expressions with this disease (Yoon et at 2006)

Recent studies stated that cancer developed in human is related to ribosomal proteins

(RPs) (Daftuar Zhu Jacq amp Prives 2013) The study using zebrafish (Danio rerio) as an

animal model has shown that several RP genes are cancer genes in this organism

2

(Amsterdam et al 2004) A recent study has shown that expression level of various RPs

was either up-regulated or down-regulated in colorectal carcinoma (CRC) (Lai amp Xu 2007)

There were several RP genes that were found to be downregulated in NPC namely

RPS26 and RPS27 (Sim Toh amp Tiong 2008) Currently there is a lack of findings on the

relationship between the gene expression of RPS15 and nasopharyngeal cancer

This research is aimed to compare the differential expression level of RPS15 gene in

normal NP69 cell line and tumour HKI cell line The RPS15 is expected to show

significant difference in gene expression in comparison between normal and NPC cell line

The study on gene expression of RPs is in the hope to bring a breakthrough in the

study of cancer Hopefully gene expression of RPs will contributes to future cancer

researches such as in biomarker assessment gene manipulation and silencing in cancer

therapy

12 Objectives

The objectives throughout the completion of this project are of the following

1 To detect the expression of RPS15 gene in NP69 normal nasopharyngeal cell line and

HKI NPC cell line

2 To compare the differential expression level of RPS15 gene in NP69 normal human

nasopharyngeal cell line and HKI NPC cell line

3

l I

20 Literature Review

21 Nasopharyngeal Carcinoma (NPC)

Zeng and Zeng (2010) mentioned that NPC is found at the Eustachian tube surrounding

ostium that is situated in the nasopharynx lateral wall This disease have a bimodal

distribution in the age group where it affects people between age of 50 to 60 and children at

late childhood NPC also found higher in men than women (Zeng amp Zeng 2010)

211 Causative Factors of NPC

The major causes of NPC are geographical and population factor environmental factor

genetic factors and meal consumption factor (Zeng amp Zeng 2010) NPC was recorded to

affect population regionally and geographically as there was a highest rates of occurrence in

Southern Chinese population in Guangdong Greelanders native and Inuits of Alaska

compared to lower occurrence among population in Caucasians from Western countries

(Zeng amp Zeng 2010) Besides genetic factor was found to be causative factor due to familial

NPC history among Cantonese population (Zeng amp Zeng 2010) The haplotypes which is

the inherited genes from a single parent ofhuman leukocyte antigen (HLA) detected among

Chinese population who were being diagnosed with NPC (Zeng amp Zeng 2010) Moreover

components inside preserved food for example N-nitrospiperidine (NPIP)

nitrosodimethyamine (NDMA) and N-nitrospyrrolidene (NPYR) are the causative agents in

developing NPC (Zeng amp Zeng 2010) Another causative factor of NPC was caused by

DNA tumour virus which was the Epstein - Barr virus (EBV) infection This is because viral

genome ofEBV was found in the carcinoma cells ofNPC (Feinmesser et ai 1992)

4

Pusat Khidmat Maklumat Akad(~v UNlVEltSm MALAYSIA SAR4WAK

212 Staging of NPC

According to Hu (2010) NPC spread easily through lymph system resulted in early lymph

node around the neck Thus NPC is highly metastatic The chance of recovery depends

greatly on the staging of the cancer whether cancer has spread through the enlargement of

tumour size involvement of lymph node and metastasis (Hu 2010) Stage I NPC is a small

tumour found regionally in the nasopharynx Stage II NPC is a tumour found to extend from

nasopharynx to the oropharynx or spread to one or more lymph nodes on the side of the neck

Stage III NPC is a large tumour that has spread to surrounding sinuses or spread to lymph

nodes on both sides ofthe neck Stage IV NPC is large tumour that spread out ofnasopharynx

region or affected lymph nodes becomes larger with distant metastasis Hu (2010) mentioned

that patients from early stage have better prognosis than patient in stage III or higher due to

distant metastasis and lymph node involvement

213 Histopathological types of NPC

Three histological subtypes of NPC have been categorized by World Health Organization

(WHO) according to differentiation of cells (Zeng amp Zeng 2010)

First Type 1 under electron microscope there is intracellular bridges found in

squamous cell carcinoma (SCC) and good cell differentiation that makes keratin (Zeng amp

Zeng 2010) Type 2 is a squamous carcinoma that does not makes keratin and have cell

differentiation varied from mature to anaplastic (Zeng amp Zeng 2010) While Type 3 the

cells types are inconsistent and variable This undifferentiated and non-keratinizing Type 3

NPC found to make up the nasopharyngeal tumours (Zeng amp Zeng 2010) Zeng and Zeng

(2010) also stated that this tumour was considered as lymphoepithelioma due to its tumour

infiltration with lymphocytes Zeng and Zeng (2010) stated that type 3 NPC are more

common in Southern China while type 1 usually found in Western countries the nonshy

5

endemic areas In Type 3 NPC it was diagnosed with the presence ofEBV transcript and the

diagnosis was based on the location of tumour in nasopharynx (Zeng amp Zeng 2010)

However there was also another classification system suggested to group NPC based

on the state ofdifferentiation of NPC Flint et al (2010) suggested that NPC was able to be

classified into different type based on the state of differentiation namely Type 1 Type 2a

and Type 2b Well differentiated SCC that was keratinizing was grouped into Type 1 There

was slightly different between Type 2a and Type 2b Flint et al (2010) mentioned that SCC

that was non-keratinizing was classified as Type 2a While the non-keratinizing carcinoma

which was in the state of undifferentiated was classified as Type 2b

214 Genes Linked to NPC

There are a few RPs gene found to be associated with NPC According to Sim et al (2008)

the RPs gene that linked to NPC wereRPS26 and RPS27 The RPS26 was downregulated in

NPC cell which was more noticeable in Type II b NPC RPS27 also found to be

downregulated in Type II a and Type II b of NPC cell types in contrast to normal

nasopharyngeal cell (Sim et al 2008)

6

22 Ribosomal Proteins (RPs)

According to Robledo et a (2008) the combination of rRNA with RPs together with

nonribosomal protein and other factors are involved in the production of ribosomes Robledo

et al (2008) stated that subunits ofmature ribosome form through the association of the RPs

with the ribosomal genes that undergo transcription in nucleolus by RNA polymerase I The

subunits produced are 40S and 60S subunit (Robledo et al 2008)

Besides the contribution ofRPs in the making up of the subunits oforganelle in cell

it was found to have the extra-ribosomal functions (Bhavsar et a 20 I 0) The extrashy

ribosomal functions are for example replication splicing transcription and regulation of

life span This special cellular functions is independent of ribosomes (Bhavsar et al 2010)

Bhavsar et al (2010) mentioned that regulation of RPs are so special that it was able to

regulate its expression level and control cell cycle and apoptosis

221 Extra-ribosomal functions of RPs

Bhavsar et a (2010) pointed out that at different levels of gene regulation RPs able to

regulate its own expression as well as of other gene RPs took part in mechanisms of gene

regulation at different levels for example modification of chromatin transcription

translation post-translation modification and RNA processing (Bhavsar et a 2010)

Besides RPs also involved in replication of viral genome either DNA or RNA

viruses during viral infection (Bhavsar et al 2010) Bhavsar et al (2010) mentioned that

RPs also involved in DNA repair mechanism when DNA is damaged by cell metabolic

processes or environmental factors in both prokaryotes and eukaryotes

Moreover RPs are involved in the cell cycle regulation by acting together with the

regulatory molecules like cyclin-dependent kinase (Cdk) According to Bhavsar et a (2010)

7

Cdk controlled the advancement of cell to different phases of cell cycle The expression

levels of RPs also regulate cell cycle by activating apoptosis (Bhavsar et al 2010)

According to Boon and Sim (2015) apoptosis is known as normal process of cell death

Bhavsar et al (2010) stated that during apoptosis RPs interact with an ubiquitin ligase

Mouse double minute 2 (Mdm2) to validate a normal p53 level

23 Ribosomal Protein S15 (RPS15)

Kenmochi Ashworth Lennon Hisa and Tanaka (1998) found that several human RPs genes

are situated in chromosome RPS 15 gene is situated in chromosome 19 in human According

to Kenmochi et al (1998) the mapped position ofRPS15 gene in chromosome 19 is 19p 133

the distance from the top of short arm is 16 Mb and distance from the most distal short-arm

marker is 6cR

ll39726~ ~ l1490888~

~--------------~~--~----------------------~ GAKI tpSlS J C190rf25

DAZAP1------~I PCSK4 -shy~(C2 --------shy

Figure 21 Circled indicates the location of RPSI5 gene in chromosome 19 of Homo sapiens (Adapted from hupwwwncbinlmnihgovgene6209)

According to Bhavsar et al (2010) RPS 15 in Ecoli found to have extra-ribosomal

properties in which it is self-translation regulated The expression of mutated genes or

unusual level of expression of ribosomal proteins are related with disease disorder RPS 15

is found to be involved in a congenital erythroid aplasia which is also known as Diamond-

Blackfan anaemia (DBA) (Bhavsar et al 2010)

8

l

(Amsterdam et al 2004) A recent study has shown that expression level of various RPs

was either up-regulated or down-regulated in colorectal carcinoma (CRC) (Lai amp Xu 2007)

There were several RP genes that were found to be downregulated in NPC namely

RPS26 and RPS27 (Sim Toh amp Tiong 2008) Currently there is a lack of findings on the

relationship between the gene expression of RPS15 and nasopharyngeal cancer

This research is aimed to compare the differential expression level of RPS15 gene in

normal NP69 cell line and tumour HKI cell line The RPS15 is expected to show

significant difference in gene expression in comparison between normal and NPC cell line

The study on gene expression of RPs is in the hope to bring a breakthrough in the

study of cancer Hopefully gene expression of RPs will contributes to future cancer

researches such as in biomarker assessment gene manipulation and silencing in cancer

therapy

12 Objectives

The objectives throughout the completion of this project are of the following

1 To detect the expression of RPS15 gene in NP69 normal nasopharyngeal cell line and

HKI NPC cell line

2 To compare the differential expression level of RPS15 gene in NP69 normal human

nasopharyngeal cell line and HKI NPC cell line

3

l I

20 Literature Review

21 Nasopharyngeal Carcinoma (NPC)

Zeng and Zeng (2010) mentioned that NPC is found at the Eustachian tube surrounding

ostium that is situated in the nasopharynx lateral wall This disease have a bimodal

distribution in the age group where it affects people between age of 50 to 60 and children at

late childhood NPC also found higher in men than women (Zeng amp Zeng 2010)

211 Causative Factors of NPC

The major causes of NPC are geographical and population factor environmental factor

genetic factors and meal consumption factor (Zeng amp Zeng 2010) NPC was recorded to

affect population regionally and geographically as there was a highest rates of occurrence in

Southern Chinese population in Guangdong Greelanders native and Inuits of Alaska

compared to lower occurrence among population in Caucasians from Western countries

(Zeng amp Zeng 2010) Besides genetic factor was found to be causative factor due to familial

NPC history among Cantonese population (Zeng amp Zeng 2010) The haplotypes which is

the inherited genes from a single parent ofhuman leukocyte antigen (HLA) detected among

Chinese population who were being diagnosed with NPC (Zeng amp Zeng 2010) Moreover

components inside preserved food for example N-nitrospiperidine (NPIP)

nitrosodimethyamine (NDMA) and N-nitrospyrrolidene (NPYR) are the causative agents in

developing NPC (Zeng amp Zeng 2010) Another causative factor of NPC was caused by

DNA tumour virus which was the Epstein - Barr virus (EBV) infection This is because viral

genome ofEBV was found in the carcinoma cells ofNPC (Feinmesser et ai 1992)

4

Pusat Khidmat Maklumat Akad(~v UNlVEltSm MALAYSIA SAR4WAK

212 Staging of NPC

According to Hu (2010) NPC spread easily through lymph system resulted in early lymph

node around the neck Thus NPC is highly metastatic The chance of recovery depends

greatly on the staging of the cancer whether cancer has spread through the enlargement of

tumour size involvement of lymph node and metastasis (Hu 2010) Stage I NPC is a small

tumour found regionally in the nasopharynx Stage II NPC is a tumour found to extend from

nasopharynx to the oropharynx or spread to one or more lymph nodes on the side of the neck

Stage III NPC is a large tumour that has spread to surrounding sinuses or spread to lymph

nodes on both sides ofthe neck Stage IV NPC is large tumour that spread out ofnasopharynx

region or affected lymph nodes becomes larger with distant metastasis Hu (2010) mentioned

that patients from early stage have better prognosis than patient in stage III or higher due to

distant metastasis and lymph node involvement

213 Histopathological types of NPC

Three histological subtypes of NPC have been categorized by World Health Organization

(WHO) according to differentiation of cells (Zeng amp Zeng 2010)

First Type 1 under electron microscope there is intracellular bridges found in

squamous cell carcinoma (SCC) and good cell differentiation that makes keratin (Zeng amp

Zeng 2010) Type 2 is a squamous carcinoma that does not makes keratin and have cell

differentiation varied from mature to anaplastic (Zeng amp Zeng 2010) While Type 3 the

cells types are inconsistent and variable This undifferentiated and non-keratinizing Type 3

NPC found to make up the nasopharyngeal tumours (Zeng amp Zeng 2010) Zeng and Zeng

(2010) also stated that this tumour was considered as lymphoepithelioma due to its tumour

infiltration with lymphocytes Zeng and Zeng (2010) stated that type 3 NPC are more

common in Southern China while type 1 usually found in Western countries the nonshy

5

endemic areas In Type 3 NPC it was diagnosed with the presence ofEBV transcript and the

diagnosis was based on the location of tumour in nasopharynx (Zeng amp Zeng 2010)

However there was also another classification system suggested to group NPC based

on the state ofdifferentiation of NPC Flint et al (2010) suggested that NPC was able to be

classified into different type based on the state of differentiation namely Type 1 Type 2a

and Type 2b Well differentiated SCC that was keratinizing was grouped into Type 1 There

was slightly different between Type 2a and Type 2b Flint et al (2010) mentioned that SCC

that was non-keratinizing was classified as Type 2a While the non-keratinizing carcinoma

which was in the state of undifferentiated was classified as Type 2b

214 Genes Linked to NPC

There are a few RPs gene found to be associated with NPC According to Sim et al (2008)

the RPs gene that linked to NPC wereRPS26 and RPS27 The RPS26 was downregulated in

NPC cell which was more noticeable in Type II b NPC RPS27 also found to be

downregulated in Type II a and Type II b of NPC cell types in contrast to normal

nasopharyngeal cell (Sim et al 2008)

6

22 Ribosomal Proteins (RPs)

According to Robledo et a (2008) the combination of rRNA with RPs together with

nonribosomal protein and other factors are involved in the production of ribosomes Robledo

et al (2008) stated that subunits ofmature ribosome form through the association of the RPs

with the ribosomal genes that undergo transcription in nucleolus by RNA polymerase I The

subunits produced are 40S and 60S subunit (Robledo et al 2008)

Besides the contribution ofRPs in the making up of the subunits oforganelle in cell

it was found to have the extra-ribosomal functions (Bhavsar et a 20 I 0) The extrashy

ribosomal functions are for example replication splicing transcription and regulation of

life span This special cellular functions is independent of ribosomes (Bhavsar et al 2010)

Bhavsar et al (2010) mentioned that regulation of RPs are so special that it was able to

regulate its expression level and control cell cycle and apoptosis

221 Extra-ribosomal functions of RPs

Bhavsar et a (2010) pointed out that at different levels of gene regulation RPs able to

regulate its own expression as well as of other gene RPs took part in mechanisms of gene

regulation at different levels for example modification of chromatin transcription

translation post-translation modification and RNA processing (Bhavsar et a 2010)

Besides RPs also involved in replication of viral genome either DNA or RNA

viruses during viral infection (Bhavsar et al 2010) Bhavsar et al (2010) mentioned that

RPs also involved in DNA repair mechanism when DNA is damaged by cell metabolic

processes or environmental factors in both prokaryotes and eukaryotes

Moreover RPs are involved in the cell cycle regulation by acting together with the

regulatory molecules like cyclin-dependent kinase (Cdk) According to Bhavsar et a (2010)

7

Cdk controlled the advancement of cell to different phases of cell cycle The expression

levels of RPs also regulate cell cycle by activating apoptosis (Bhavsar et al 2010)

According to Boon and Sim (2015) apoptosis is known as normal process of cell death

Bhavsar et al (2010) stated that during apoptosis RPs interact with an ubiquitin ligase

Mouse double minute 2 (Mdm2) to validate a normal p53 level

23 Ribosomal Protein S15 (RPS15)

Kenmochi Ashworth Lennon Hisa and Tanaka (1998) found that several human RPs genes

are situated in chromosome RPS 15 gene is situated in chromosome 19 in human According

to Kenmochi et al (1998) the mapped position ofRPS15 gene in chromosome 19 is 19p 133

the distance from the top of short arm is 16 Mb and distance from the most distal short-arm

marker is 6cR

ll39726~ ~ l1490888~

~--------------~~--~----------------------~ GAKI tpSlS J C190rf25

DAZAP1------~I PCSK4 -shy~(C2 --------shy

Figure 21 Circled indicates the location of RPSI5 gene in chromosome 19 of Homo sapiens (Adapted from hupwwwncbinlmnihgovgene6209)

According to Bhavsar et al (2010) RPS 15 in Ecoli found to have extra-ribosomal

properties in which it is self-translation regulated The expression of mutated genes or

unusual level of expression of ribosomal proteins are related with disease disorder RPS 15

is found to be involved in a congenital erythroid aplasia which is also known as Diamond-

Blackfan anaemia (DBA) (Bhavsar et al 2010)

8

l

20 Literature Review

21 Nasopharyngeal Carcinoma (NPC)

Zeng and Zeng (2010) mentioned that NPC is found at the Eustachian tube surrounding

ostium that is situated in the nasopharynx lateral wall This disease have a bimodal

distribution in the age group where it affects people between age of 50 to 60 and children at

late childhood NPC also found higher in men than women (Zeng amp Zeng 2010)

211 Causative Factors of NPC

The major causes of NPC are geographical and population factor environmental factor

genetic factors and meal consumption factor (Zeng amp Zeng 2010) NPC was recorded to

affect population regionally and geographically as there was a highest rates of occurrence in

Southern Chinese population in Guangdong Greelanders native and Inuits of Alaska

compared to lower occurrence among population in Caucasians from Western countries

(Zeng amp Zeng 2010) Besides genetic factor was found to be causative factor due to familial

NPC history among Cantonese population (Zeng amp Zeng 2010) The haplotypes which is

the inherited genes from a single parent ofhuman leukocyte antigen (HLA) detected among

Chinese population who were being diagnosed with NPC (Zeng amp Zeng 2010) Moreover

components inside preserved food for example N-nitrospiperidine (NPIP)

nitrosodimethyamine (NDMA) and N-nitrospyrrolidene (NPYR) are the causative agents in

developing NPC (Zeng amp Zeng 2010) Another causative factor of NPC was caused by

DNA tumour virus which was the Epstein - Barr virus (EBV) infection This is because viral

genome ofEBV was found in the carcinoma cells ofNPC (Feinmesser et ai 1992)

4

Pusat Khidmat Maklumat Akad(~v UNlVEltSm MALAYSIA SAR4WAK

212 Staging of NPC

According to Hu (2010) NPC spread easily through lymph system resulted in early lymph

node around the neck Thus NPC is highly metastatic The chance of recovery depends

greatly on the staging of the cancer whether cancer has spread through the enlargement of

tumour size involvement of lymph node and metastasis (Hu 2010) Stage I NPC is a small

tumour found regionally in the nasopharynx Stage II NPC is a tumour found to extend from

nasopharynx to the oropharynx or spread to one or more lymph nodes on the side of the neck

Stage III NPC is a large tumour that has spread to surrounding sinuses or spread to lymph

nodes on both sides ofthe neck Stage IV NPC is large tumour that spread out ofnasopharynx

region or affected lymph nodes becomes larger with distant metastasis Hu (2010) mentioned

that patients from early stage have better prognosis than patient in stage III or higher due to

distant metastasis and lymph node involvement

213 Histopathological types of NPC

Three histological subtypes of NPC have been categorized by World Health Organization

(WHO) according to differentiation of cells (Zeng amp Zeng 2010)

First Type 1 under electron microscope there is intracellular bridges found in

squamous cell carcinoma (SCC) and good cell differentiation that makes keratin (Zeng amp

Zeng 2010) Type 2 is a squamous carcinoma that does not makes keratin and have cell

differentiation varied from mature to anaplastic (Zeng amp Zeng 2010) While Type 3 the

cells types are inconsistent and variable This undifferentiated and non-keratinizing Type 3

NPC found to make up the nasopharyngeal tumours (Zeng amp Zeng 2010) Zeng and Zeng

(2010) also stated that this tumour was considered as lymphoepithelioma due to its tumour

infiltration with lymphocytes Zeng and Zeng (2010) stated that type 3 NPC are more

common in Southern China while type 1 usually found in Western countries the nonshy

5

endemic areas In Type 3 NPC it was diagnosed with the presence ofEBV transcript and the

diagnosis was based on the location of tumour in nasopharynx (Zeng amp Zeng 2010)

However there was also another classification system suggested to group NPC based

on the state ofdifferentiation of NPC Flint et al (2010) suggested that NPC was able to be

classified into different type based on the state of differentiation namely Type 1 Type 2a

and Type 2b Well differentiated SCC that was keratinizing was grouped into Type 1 There

was slightly different between Type 2a and Type 2b Flint et al (2010) mentioned that SCC

that was non-keratinizing was classified as Type 2a While the non-keratinizing carcinoma

which was in the state of undifferentiated was classified as Type 2b

214 Genes Linked to NPC

There are a few RPs gene found to be associated with NPC According to Sim et al (2008)

the RPs gene that linked to NPC wereRPS26 and RPS27 The RPS26 was downregulated in

NPC cell which was more noticeable in Type II b NPC RPS27 also found to be

downregulated in Type II a and Type II b of NPC cell types in contrast to normal

nasopharyngeal cell (Sim et al 2008)

6

22 Ribosomal Proteins (RPs)

According to Robledo et a (2008) the combination of rRNA with RPs together with

nonribosomal protein and other factors are involved in the production of ribosomes Robledo

et al (2008) stated that subunits ofmature ribosome form through the association of the RPs

with the ribosomal genes that undergo transcription in nucleolus by RNA polymerase I The

subunits produced are 40S and 60S subunit (Robledo et al 2008)

Besides the contribution ofRPs in the making up of the subunits oforganelle in cell

it was found to have the extra-ribosomal functions (Bhavsar et a 20 I 0) The extrashy

ribosomal functions are for example replication splicing transcription and regulation of

life span This special cellular functions is independent of ribosomes (Bhavsar et al 2010)

Bhavsar et al (2010) mentioned that regulation of RPs are so special that it was able to

regulate its expression level and control cell cycle and apoptosis

221 Extra-ribosomal functions of RPs

Bhavsar et a (2010) pointed out that at different levels of gene regulation RPs able to

regulate its own expression as well as of other gene RPs took part in mechanisms of gene

regulation at different levels for example modification of chromatin transcription

translation post-translation modification and RNA processing (Bhavsar et a 2010)

Besides RPs also involved in replication of viral genome either DNA or RNA

viruses during viral infection (Bhavsar et al 2010) Bhavsar et al (2010) mentioned that

RPs also involved in DNA repair mechanism when DNA is damaged by cell metabolic

processes or environmental factors in both prokaryotes and eukaryotes

Moreover RPs are involved in the cell cycle regulation by acting together with the

regulatory molecules like cyclin-dependent kinase (Cdk) According to Bhavsar et a (2010)

7

Cdk controlled the advancement of cell to different phases of cell cycle The expression

levels of RPs also regulate cell cycle by activating apoptosis (Bhavsar et al 2010)

According to Boon and Sim (2015) apoptosis is known as normal process of cell death

Bhavsar et al (2010) stated that during apoptosis RPs interact with an ubiquitin ligase

Mouse double minute 2 (Mdm2) to validate a normal p53 level

23 Ribosomal Protein S15 (RPS15)

Kenmochi Ashworth Lennon Hisa and Tanaka (1998) found that several human RPs genes

are situated in chromosome RPS 15 gene is situated in chromosome 19 in human According

to Kenmochi et al (1998) the mapped position ofRPS15 gene in chromosome 19 is 19p 133

the distance from the top of short arm is 16 Mb and distance from the most distal short-arm

marker is 6cR

ll39726~ ~ l1490888~

~--------------~~--~----------------------~ GAKI tpSlS J C190rf25

DAZAP1------~I PCSK4 -shy~(C2 --------shy

Figure 21 Circled indicates the location of RPSI5 gene in chromosome 19 of Homo sapiens (Adapted from hupwwwncbinlmnihgovgene6209)

According to Bhavsar et al (2010) RPS 15 in Ecoli found to have extra-ribosomal

properties in which it is self-translation regulated The expression of mutated genes or

unusual level of expression of ribosomal proteins are related with disease disorder RPS 15

is found to be involved in a congenital erythroid aplasia which is also known as Diamond-

Blackfan anaemia (DBA) (Bhavsar et al 2010)

8

l

Pusat Khidmat Maklumat Akad(~v UNlVEltSm MALAYSIA SAR4WAK

212 Staging of NPC

According to Hu (2010) NPC spread easily through lymph system resulted in early lymph

node around the neck Thus NPC is highly metastatic The chance of recovery depends

greatly on the staging of the cancer whether cancer has spread through the enlargement of

tumour size involvement of lymph node and metastasis (Hu 2010) Stage I NPC is a small

tumour found regionally in the nasopharynx Stage II NPC is a tumour found to extend from

nasopharynx to the oropharynx or spread to one or more lymph nodes on the side of the neck

Stage III NPC is a large tumour that has spread to surrounding sinuses or spread to lymph

nodes on both sides ofthe neck Stage IV NPC is large tumour that spread out ofnasopharynx

region or affected lymph nodes becomes larger with distant metastasis Hu (2010) mentioned

that patients from early stage have better prognosis than patient in stage III or higher due to

distant metastasis and lymph node involvement

213 Histopathological types of NPC

Three histological subtypes of NPC have been categorized by World Health Organization

(WHO) according to differentiation of cells (Zeng amp Zeng 2010)

First Type 1 under electron microscope there is intracellular bridges found in

squamous cell carcinoma (SCC) and good cell differentiation that makes keratin (Zeng amp

Zeng 2010) Type 2 is a squamous carcinoma that does not makes keratin and have cell

differentiation varied from mature to anaplastic (Zeng amp Zeng 2010) While Type 3 the

cells types are inconsistent and variable This undifferentiated and non-keratinizing Type 3

NPC found to make up the nasopharyngeal tumours (Zeng amp Zeng 2010) Zeng and Zeng

(2010) also stated that this tumour was considered as lymphoepithelioma due to its tumour

infiltration with lymphocytes Zeng and Zeng (2010) stated that type 3 NPC are more

common in Southern China while type 1 usually found in Western countries the nonshy

5

endemic areas In Type 3 NPC it was diagnosed with the presence ofEBV transcript and the

diagnosis was based on the location of tumour in nasopharynx (Zeng amp Zeng 2010)

However there was also another classification system suggested to group NPC based

on the state ofdifferentiation of NPC Flint et al (2010) suggested that NPC was able to be

classified into different type based on the state of differentiation namely Type 1 Type 2a

and Type 2b Well differentiated SCC that was keratinizing was grouped into Type 1 There

was slightly different between Type 2a and Type 2b Flint et al (2010) mentioned that SCC

that was non-keratinizing was classified as Type 2a While the non-keratinizing carcinoma

which was in the state of undifferentiated was classified as Type 2b

214 Genes Linked to NPC

There are a few RPs gene found to be associated with NPC According to Sim et al (2008)

the RPs gene that linked to NPC wereRPS26 and RPS27 The RPS26 was downregulated in

NPC cell which was more noticeable in Type II b NPC RPS27 also found to be

downregulated in Type II a and Type II b of NPC cell types in contrast to normal

nasopharyngeal cell (Sim et al 2008)

6

22 Ribosomal Proteins (RPs)

According to Robledo et a (2008) the combination of rRNA with RPs together with

nonribosomal protein and other factors are involved in the production of ribosomes Robledo

et al (2008) stated that subunits ofmature ribosome form through the association of the RPs

with the ribosomal genes that undergo transcription in nucleolus by RNA polymerase I The

subunits produced are 40S and 60S subunit (Robledo et al 2008)

Besides the contribution ofRPs in the making up of the subunits oforganelle in cell

it was found to have the extra-ribosomal functions (Bhavsar et a 20 I 0) The extrashy

ribosomal functions are for example replication splicing transcription and regulation of

life span This special cellular functions is independent of ribosomes (Bhavsar et al 2010)

Bhavsar et al (2010) mentioned that regulation of RPs are so special that it was able to

regulate its expression level and control cell cycle and apoptosis

221 Extra-ribosomal functions of RPs

Bhavsar et a (2010) pointed out that at different levels of gene regulation RPs able to

regulate its own expression as well as of other gene RPs took part in mechanisms of gene

regulation at different levels for example modification of chromatin transcription

translation post-translation modification and RNA processing (Bhavsar et a 2010)

Besides RPs also involved in replication of viral genome either DNA or RNA

viruses during viral infection (Bhavsar et al 2010) Bhavsar et al (2010) mentioned that

RPs also involved in DNA repair mechanism when DNA is damaged by cell metabolic

processes or environmental factors in both prokaryotes and eukaryotes

Moreover RPs are involved in the cell cycle regulation by acting together with the

regulatory molecules like cyclin-dependent kinase (Cdk) According to Bhavsar et a (2010)

7

Cdk controlled the advancement of cell to different phases of cell cycle The expression

levels of RPs also regulate cell cycle by activating apoptosis (Bhavsar et al 2010)

According to Boon and Sim (2015) apoptosis is known as normal process of cell death

Bhavsar et al (2010) stated that during apoptosis RPs interact with an ubiquitin ligase

Mouse double minute 2 (Mdm2) to validate a normal p53 level

23 Ribosomal Protein S15 (RPS15)

Kenmochi Ashworth Lennon Hisa and Tanaka (1998) found that several human RPs genes

are situated in chromosome RPS 15 gene is situated in chromosome 19 in human According

to Kenmochi et al (1998) the mapped position ofRPS15 gene in chromosome 19 is 19p 133

the distance from the top of short arm is 16 Mb and distance from the most distal short-arm

marker is 6cR

ll39726~ ~ l1490888~

~--------------~~--~----------------------~ GAKI tpSlS J C190rf25

DAZAP1------~I PCSK4 -shy~(C2 --------shy

Figure 21 Circled indicates the location of RPSI5 gene in chromosome 19 of Homo sapiens (Adapted from hupwwwncbinlmnihgovgene6209)

According to Bhavsar et al (2010) RPS 15 in Ecoli found to have extra-ribosomal

properties in which it is self-translation regulated The expression of mutated genes or

unusual level of expression of ribosomal proteins are related with disease disorder RPS 15

is found to be involved in a congenital erythroid aplasia which is also known as Diamond-

Blackfan anaemia (DBA) (Bhavsar et al 2010)

8

l

endemic areas In Type 3 NPC it was diagnosed with the presence ofEBV transcript and the

diagnosis was based on the location of tumour in nasopharynx (Zeng amp Zeng 2010)

However there was also another classification system suggested to group NPC based

on the state ofdifferentiation of NPC Flint et al (2010) suggested that NPC was able to be

classified into different type based on the state of differentiation namely Type 1 Type 2a

and Type 2b Well differentiated SCC that was keratinizing was grouped into Type 1 There

was slightly different between Type 2a and Type 2b Flint et al (2010) mentioned that SCC

that was non-keratinizing was classified as Type 2a While the non-keratinizing carcinoma

which was in the state of undifferentiated was classified as Type 2b

214 Genes Linked to NPC

There are a few RPs gene found to be associated with NPC According to Sim et al (2008)

the RPs gene that linked to NPC wereRPS26 and RPS27 The RPS26 was downregulated in

NPC cell which was more noticeable in Type II b NPC RPS27 also found to be

downregulated in Type II a and Type II b of NPC cell types in contrast to normal

nasopharyngeal cell (Sim et al 2008)

6

22 Ribosomal Proteins (RPs)

According to Robledo et a (2008) the combination of rRNA with RPs together with

nonribosomal protein and other factors are involved in the production of ribosomes Robledo

et al (2008) stated that subunits ofmature ribosome form through the association of the RPs

with the ribosomal genes that undergo transcription in nucleolus by RNA polymerase I The

subunits produced are 40S and 60S subunit (Robledo et al 2008)

Besides the contribution ofRPs in the making up of the subunits oforganelle in cell

it was found to have the extra-ribosomal functions (Bhavsar et a 20 I 0) The extrashy

ribosomal functions are for example replication splicing transcription and regulation of

life span This special cellular functions is independent of ribosomes (Bhavsar et al 2010)

Bhavsar et al (2010) mentioned that regulation of RPs are so special that it was able to

regulate its expression level and control cell cycle and apoptosis

221 Extra-ribosomal functions of RPs

Bhavsar et a (2010) pointed out that at different levels of gene regulation RPs able to

regulate its own expression as well as of other gene RPs took part in mechanisms of gene

regulation at different levels for example modification of chromatin transcription

translation post-translation modification and RNA processing (Bhavsar et a 2010)

Besides RPs also involved in replication of viral genome either DNA or RNA

viruses during viral infection (Bhavsar et al 2010) Bhavsar et al (2010) mentioned that

RPs also involved in DNA repair mechanism when DNA is damaged by cell metabolic

processes or environmental factors in both prokaryotes and eukaryotes

Moreover RPs are involved in the cell cycle regulation by acting together with the

regulatory molecules like cyclin-dependent kinase (Cdk) According to Bhavsar et a (2010)

7

Cdk controlled the advancement of cell to different phases of cell cycle The expression

levels of RPs also regulate cell cycle by activating apoptosis (Bhavsar et al 2010)

According to Boon and Sim (2015) apoptosis is known as normal process of cell death

Bhavsar et al (2010) stated that during apoptosis RPs interact with an ubiquitin ligase

Mouse double minute 2 (Mdm2) to validate a normal p53 level

23 Ribosomal Protein S15 (RPS15)

Kenmochi Ashworth Lennon Hisa and Tanaka (1998) found that several human RPs genes

are situated in chromosome RPS 15 gene is situated in chromosome 19 in human According

to Kenmochi et al (1998) the mapped position ofRPS15 gene in chromosome 19 is 19p 133

the distance from the top of short arm is 16 Mb and distance from the most distal short-arm

marker is 6cR

ll39726~ ~ l1490888~

~--------------~~--~----------------------~ GAKI tpSlS J C190rf25

DAZAP1------~I PCSK4 -shy~(C2 --------shy

Figure 21 Circled indicates the location of RPSI5 gene in chromosome 19 of Homo sapiens (Adapted from hupwwwncbinlmnihgovgene6209)

According to Bhavsar et al (2010) RPS 15 in Ecoli found to have extra-ribosomal

properties in which it is self-translation regulated The expression of mutated genes or

unusual level of expression of ribosomal proteins are related with disease disorder RPS 15

is found to be involved in a congenital erythroid aplasia which is also known as Diamond-

Blackfan anaemia (DBA) (Bhavsar et al 2010)

8

l

22 Ribosomal Proteins (RPs)

According to Robledo et a (2008) the combination of rRNA with RPs together with

nonribosomal protein and other factors are involved in the production of ribosomes Robledo

et al (2008) stated that subunits ofmature ribosome form through the association of the RPs

with the ribosomal genes that undergo transcription in nucleolus by RNA polymerase I The

subunits produced are 40S and 60S subunit (Robledo et al 2008)

Besides the contribution ofRPs in the making up of the subunits oforganelle in cell

it was found to have the extra-ribosomal functions (Bhavsar et a 20 I 0) The extrashy

ribosomal functions are for example replication splicing transcription and regulation of

life span This special cellular functions is independent of ribosomes (Bhavsar et al 2010)

Bhavsar et al (2010) mentioned that regulation of RPs are so special that it was able to

regulate its expression level and control cell cycle and apoptosis

221 Extra-ribosomal functions of RPs

Bhavsar et a (2010) pointed out that at different levels of gene regulation RPs able to

regulate its own expression as well as of other gene RPs took part in mechanisms of gene

regulation at different levels for example modification of chromatin transcription

translation post-translation modification and RNA processing (Bhavsar et a 2010)

Besides RPs also involved in replication of viral genome either DNA or RNA

viruses during viral infection (Bhavsar et al 2010) Bhavsar et al (2010) mentioned that

RPs also involved in DNA repair mechanism when DNA is damaged by cell metabolic

processes or environmental factors in both prokaryotes and eukaryotes

Moreover RPs are involved in the cell cycle regulation by acting together with the

regulatory molecules like cyclin-dependent kinase (Cdk) According to Bhavsar et a (2010)

7

Cdk controlled the advancement of cell to different phases of cell cycle The expression

levels of RPs also regulate cell cycle by activating apoptosis (Bhavsar et al 2010)

According to Boon and Sim (2015) apoptosis is known as normal process of cell death

Bhavsar et al (2010) stated that during apoptosis RPs interact with an ubiquitin ligase

Mouse double minute 2 (Mdm2) to validate a normal p53 level

23 Ribosomal Protein S15 (RPS15)

Kenmochi Ashworth Lennon Hisa and Tanaka (1998) found that several human RPs genes

are situated in chromosome RPS 15 gene is situated in chromosome 19 in human According

to Kenmochi et al (1998) the mapped position ofRPS15 gene in chromosome 19 is 19p 133

the distance from the top of short arm is 16 Mb and distance from the most distal short-arm

marker is 6cR

ll39726~ ~ l1490888~

~--------------~~--~----------------------~ GAKI tpSlS J C190rf25

DAZAP1------~I PCSK4 -shy~(C2 --------shy

Figure 21 Circled indicates the location of RPSI5 gene in chromosome 19 of Homo sapiens (Adapted from hupwwwncbinlmnihgovgene6209)

According to Bhavsar et al (2010) RPS 15 in Ecoli found to have extra-ribosomal

properties in which it is self-translation regulated The expression of mutated genes or

unusual level of expression of ribosomal proteins are related with disease disorder RPS 15

is found to be involved in a congenital erythroid aplasia which is also known as Diamond-

Blackfan anaemia (DBA) (Bhavsar et al 2010)

8

l

Cdk controlled the advancement of cell to different phases of cell cycle The expression

levels of RPs also regulate cell cycle by activating apoptosis (Bhavsar et al 2010)

According to Boon and Sim (2015) apoptosis is known as normal process of cell death

Bhavsar et al (2010) stated that during apoptosis RPs interact with an ubiquitin ligase

Mouse double minute 2 (Mdm2) to validate a normal p53 level

23 Ribosomal Protein S15 (RPS15)

Kenmochi Ashworth Lennon Hisa and Tanaka (1998) found that several human RPs genes

are situated in chromosome RPS 15 gene is situated in chromosome 19 in human According

to Kenmochi et al (1998) the mapped position ofRPS15 gene in chromosome 19 is 19p 133

the distance from the top of short arm is 16 Mb and distance from the most distal short-arm

marker is 6cR

ll39726~ ~ l1490888~

~--------------~~--~----------------------~ GAKI tpSlS J C190rf25

DAZAP1------~I PCSK4 -shy~(C2 --------shy

Figure 21 Circled indicates the location of RPSI5 gene in chromosome 19 of Homo sapiens (Adapted from hupwwwncbinlmnihgovgene6209)

According to Bhavsar et al (2010) RPS 15 in Ecoli found to have extra-ribosomal

properties in which it is self-translation regulated The expression of mutated genes or

unusual level of expression of ribosomal proteins are related with disease disorder RPS 15

is found to be involved in a congenital erythroid aplasia which is also known as Diamond-

Blackfan anaemia (DBA) (Bhavsar et al 2010)

8

l

231 The Relevance of RPS15 with Various Hematologic Disorders

2311 Mutation of RPs caused ribosomopathies

The defects in function and biogenesis of ribosome resulted in ribosomopathies Mutations

in the ribosomal genes impaired erythropoiesis The clinical syndromes identified is DBA

which is a hereditary anaemia which the erythroblasts constituent not more than 5 of

nucleated cell in bone marrow (Shenoy et al 2012) RPS 19 and RPS24 mutation impaired

the processing of pre-RNA in 18S RNA which decreases the production of 40S ribosomal

subunit and mature 80S ribosomes Mutation in RPS 15 gene was found in DBA (Shenoy et

al 2012) Fankhauser Cima Wild amp Krek (2012) mentioned that mutation initiated the

changes in the expression of gene involved in cancer

2312 Interaction of RPs with p53

Daftaur et al (2013) found that Mdm2 able to inhibit the degradation ofp53 The RPS15

interact with Mdm2 through N-terminal and C-terminal which is the location of p53shy

interacting domain and Really Interesting New Gene (RING) domain respectively The

ectopic expression of RPS15 RPS20 and RPL37 able to inhibit auto-ubiquitination of

Mdm2 and degradation ofp53 mediated by Mdm2 Daftaur et al (2013) mentioned that the

half-life of p53 is lengthened by RPS 15 and RPS20 Moreover overexpression of

aforementioned RPs able to assist in programmed cell death RPS 15 causes cells to arrest in

G2 phase which is mediated by p21 (Daftaur et al 2013)

Stabilization of p53 by RPs in ribosomal stress increase the expression of p21 genes

that involved in cell cycle arrest or apoptosis Shenoy et al (2012) mentioned that the

association of RPs to MDM2 causes the MDM2 mediated p53 ubiquitination and

d ation to be blocked As a result arrested the cell cycle at the same time inhibit the

9

cell division and cell growth is not regulated RPS 15 also involved in downregulation of

MdrnX protein levels as it has low affinity to the N-terminal domain and RING domain

Besides the knockdown ofRPS15 RPS20 and RPL37 by siRNA resulted in upregulation

ofp53 and p21 while downregulates the MdmX Shenoy et al (2012) pointed out that RPs

play an important role in carcinogenesis in which it has extraribosomal functions in

regulating p53 tumour suppressor pathway

24 CeU Lines Used in RPS15 Gene Expression Level Analysis

241 NP69 ceU line

According to the findings by Chan et al (2008) NP cell line (NP69) is a normal cell line as

the annexin II and ~2-tubulin expression level is higher The down regulation of annexin II

and In-tubulin expression is related with the metastasis ofthe lymph node (Chan et al 2008)

Lo et al (2003) stated that NP69 cell line is characterised as premalignant nasopharyngeal

epithelial cells as it was recognized from primary non-malignant nasopharyngeal epithelial

cells According to Tsang et al (2010) the primary cultures ofepithelial cells are recognised

as normal nasopharyngeal epithelial cells

Glaser et al (1989) supported that the normal human epithelial cells are attackable

by virus The survivability of normal epithelial cells in soft agar during transformation

causes the cells to be infected with Epstein - Barr virus (EBV) (Glaser et al 1989) Lo et al

(2003) identified that EBV-encoded latent membrane protein 1 (LMPl) causes changes in

phenotype of NP69 cells The phenotypic changes are morphologic transformation

increased cell proliferation and migration anchorage-independent growth and invasion in

premalignant nasopharyngeal epithelial cells After certain survival limit normal cells will

go replicative senescence that prevents abnormal growth of cells (Kong Guan Guo

amp Zeng 2009)

]Q

242 NPCIHKl cell line

There were a numbers of malignant nasopharyngeal cell lines found in China and Hong

Kong (Glaser et ai 1989) The NPC cell lines identified in Hong Kong was NPCHKl

(Huang et at 1980 Glaser et ai 1989) HKl cell line is a well-differentiated squamous

carcinoma which have the same morphology with the human tumour derived from male

Chinese patient (Huang et at 1980) Huang et ai (1980) confinned the HK 1 as disease cell

line through the tumours developed at the site of inoculation in athymic nude BALBc (nunu)

mice This is a type of mice missing thymus gland it has no T cells which facilitate the study

oftumour

25 Analysis of Gene Expression

Barber Hanner Coleman amp Clark (2005) stated that analysis of gene expression IS

conducted in the situation that genes from different types of tissue need to be compared In

this study differential expression of RPS15 gene is compared between nonnal and tumour

cells

251 Housekeeping gene as a control

The differential gene expression is detected by the using the techniques ofpolymerase chain

reaction (PCR) The quantity of the RNA used in batch-to-batch variations in PCR reagents

need to be controlled (Barber et ai 2005) Barber et at (2005) stated that to nonnalize the

gene expression data housekeeping gene is used as a control In this study glyceraldehydeshy

3-phosphate dehydrogenase is utilized as a control Kozera and Rapacz (2013) mentioned

that a housekeeping gene have to conserve its expression level

11

1511 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene

GAPDH gene is assigned as a control because it controls for a nonnal glycolysis pathway in

normal cell lines The GAPDH gene found in tumour cell lines have an uncontrolled

glycolysis and cell growth GAPDH gene is used as a nonnal indicator in the gene expression

analysis

16 Principles of Methodology

161 The Advantages of TRIzol Reagent

The total RNA extracted using TRlzol reagent is not contaminated with DNA and protein

(Ma Young amp SheD 2008) Ma et al (2008) stated the process of total RNA isolation also

achievable within an hour Ahmann et al (2008) mentioned that the use of TRlzol reagent

capable in obtaining variety of genetic material for example RNA DNA and protein

Besides TRlzol reagent have a powerful benefits in which it is able to process large

amount of sample where a lot of high quality RNA is obtained using a large amount of

plasma cells (Ahmann et al 2008)

12

262 Qualitative and Quantitative Analysis of RNA

The commonly used method to determine the integrity of total RNA extracted is AGE The

band intensities of ribosomal bands indicates the integrity of the total RNA A completely

intact RNA was observed to have two distinct and clear bands which corresponded to 28S

rRNA band and 18S rRNA band The extracted RNA that shown its 28S ribosomal band

intensity doubled that of 18S ribosomal band is considered intact RNA ofhigh quality where

the ratio of 28S to 18S was approximately 2 1 (Imbeaud et al 2005) According to

Wieczorek Delauriere and Schagat (2012) the functions of AGE not only determine the

integrity ofthe RNA it also able to detect the degradation ofRNA Strand Enell Hedenfalk

and Ferno (2007) mentioned that analysis of gene expression was influenced by the quality

of RNA specifically the degradation in its quality The concentration of RNA was usually

quantified by using UV-spectrophotometer (UV-spec) During the quantification process

absorbance at various wavelength were detected According to Imbeaud et al (2005) RNA

which is truly pure was determined to record a value of more than or equal to 18 in the

absorbance ratio at wavelength 260run to 280run (A160A28IJ) Apart from that pure RNA was

considered to have same values in the ratios A260A23o and A260A18o (lmbeaud et ai 2005)

But the checking of purity of RNA using UV spectrophotometer was not really sensitive as

it is easily affected by presence of contaminants (Strand et ai 2007) Over estimation of

concentration ofRNA caused by the absorbance at 260run indicated the presence ofgenomic

DNA (Imbeaud et ai 2005) While the absorbance at 280run signified the presence of

protein but not totally neglected the presence of other potential organic contaminants which

also interfered with the measurement (lmbeaud et ai 2005)

13

163 Considerations on Designing Primer

The amplification of genes of interest were accomplished by the activities of primers In

Older to observe the expression of specific gene in the sample primers were designed to

blrget the gene ofinterest The primer usuall y came in pair which are the forward and reverse

primers The primer pairs that were designed amplify intended target only According to Ye

et aI (2012) primer pairs which merely amplify the intended target were considered as

specific primer This is because the primer pairs have no any matches to other unintended

target in other words the primers were unable to amplify any undesired product by means

ofchanging its orientation or in any distances between the primer pairs (Ye et al 2012) In

the process of designing primers there were severa~ conditions that required to take into

consideration Ye et al (2012) stated that the characteristics of primer pairs includes small

difference in melting temperatures (Tm) of the primers a considerable GC content and

minimal homodimer heterodimer and hairpin structure Ye et al (2012) also pointed out

that a minimal base changes as few as possible or less than two base changes at the 3 end

allowed certain amplification efficiency Base mismatches in 5 end was more tolerable than

3 end base mismatches Ye et al (2012) stated that the primer have to be designed to flank

the region of interest

According to Gubelmann et al (2011) in the case ofthe gene comprised of transcript

variants the primers were designed to be either specific to the gene which is flanking the

common region of the variants or specific to the transcript which is covering one of the

splice variants The amplification of genomic DNA was prevented if the one of the primer

pair span the exon of the transcript (Gubelmann et al 2011) Moreover Ye et al (2012)

also stated that the amplification ofundesired genomic DNA able to be avoided with the one

primers span on one of the exon-exon junctions of the transcript variants

14

cell division and cell growth is not regulated RPS 15 also involved in downregulation of

MdrnX protein levels as it has low affinity to the N-terminal domain and RING domain

Besides the knockdown ofRPS15 RPS20 and RPL37 by siRNA resulted in upregulation

ofp53 and p21 while downregulates the MdmX Shenoy et al (2012) pointed out that RPs

play an important role in carcinogenesis in which it has extraribosomal functions in

regulating p53 tumour suppressor pathway

24 CeU Lines Used in RPS15 Gene Expression Level Analysis

241 NP69 ceU line

According to the findings by Chan et al (2008) NP cell line (NP69) is a normal cell line as

the annexin II and ~2-tubulin expression level is higher The down regulation of annexin II

and In-tubulin expression is related with the metastasis ofthe lymph node (Chan et al 2008)

Lo et al (2003) stated that NP69 cell line is characterised as premalignant nasopharyngeal

epithelial cells as it was recognized from primary non-malignant nasopharyngeal epithelial

cells According to Tsang et al (2010) the primary cultures ofepithelial cells are recognised

as normal nasopharyngeal epithelial cells

Glaser et al (1989) supported that the normal human epithelial cells are attackable

by virus The survivability of normal epithelial cells in soft agar during transformation

causes the cells to be infected with Epstein - Barr virus (EBV) (Glaser et al 1989) Lo et al

(2003) identified that EBV-encoded latent membrane protein 1 (LMPl) causes changes in

phenotype of NP69 cells The phenotypic changes are morphologic transformation

increased cell proliferation and migration anchorage-independent growth and invasion in

premalignant nasopharyngeal epithelial cells After certain survival limit normal cells will

go replicative senescence that prevents abnormal growth of cells (Kong Guan Guo

amp Zeng 2009)

]Q

242 NPCIHKl cell line

There were a numbers of malignant nasopharyngeal cell lines found in China and Hong

Kong (Glaser et ai 1989) The NPC cell lines identified in Hong Kong was NPCHKl

(Huang et at 1980 Glaser et ai 1989) HKl cell line is a well-differentiated squamous

carcinoma which have the same morphology with the human tumour derived from male

Chinese patient (Huang et at 1980) Huang et ai (1980) confinned the HK 1 as disease cell

line through the tumours developed at the site of inoculation in athymic nude BALBc (nunu)

mice This is a type of mice missing thymus gland it has no T cells which facilitate the study

oftumour

25 Analysis of Gene Expression

Barber Hanner Coleman amp Clark (2005) stated that analysis of gene expression IS

conducted in the situation that genes from different types of tissue need to be compared In

this study differential expression of RPS15 gene is compared between nonnal and tumour

cells

251 Housekeeping gene as a control

The differential gene expression is detected by the using the techniques ofpolymerase chain

reaction (PCR) The quantity of the RNA used in batch-to-batch variations in PCR reagents

need to be controlled (Barber et ai 2005) Barber et at (2005) stated that to nonnalize the

gene expression data housekeeping gene is used as a control In this study glyceraldehydeshy

3-phosphate dehydrogenase is utilized as a control Kozera and Rapacz (2013) mentioned

that a housekeeping gene have to conserve its expression level

11

1511 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene

GAPDH gene is assigned as a control because it controls for a nonnal glycolysis pathway in

normal cell lines The GAPDH gene found in tumour cell lines have an uncontrolled

glycolysis and cell growth GAPDH gene is used as a nonnal indicator in the gene expression

analysis

16 Principles of Methodology

161 The Advantages of TRIzol Reagent

The total RNA extracted using TRlzol reagent is not contaminated with DNA and protein

(Ma Young amp SheD 2008) Ma et al (2008) stated the process of total RNA isolation also

achievable within an hour Ahmann et al (2008) mentioned that the use of TRlzol reagent

capable in obtaining variety of genetic material for example RNA DNA and protein

Besides TRlzol reagent have a powerful benefits in which it is able to process large

amount of sample where a lot of high quality RNA is obtained using a large amount of

plasma cells (Ahmann et al 2008)

12

262 Qualitative and Quantitative Analysis of RNA

The commonly used method to determine the integrity of total RNA extracted is AGE The

band intensities of ribosomal bands indicates the integrity of the total RNA A completely

intact RNA was observed to have two distinct and clear bands which corresponded to 28S

rRNA band and 18S rRNA band The extracted RNA that shown its 28S ribosomal band

intensity doubled that of 18S ribosomal band is considered intact RNA ofhigh quality where

the ratio of 28S to 18S was approximately 2 1 (Imbeaud et al 2005) According to

Wieczorek Delauriere and Schagat (2012) the functions of AGE not only determine the

integrity ofthe RNA it also able to detect the degradation ofRNA Strand Enell Hedenfalk

and Ferno (2007) mentioned that analysis of gene expression was influenced by the quality

of RNA specifically the degradation in its quality The concentration of RNA was usually

quantified by using UV-spectrophotometer (UV-spec) During the quantification process

absorbance at various wavelength were detected According to Imbeaud et al (2005) RNA

which is truly pure was determined to record a value of more than or equal to 18 in the

absorbance ratio at wavelength 260run to 280run (A160A28IJ) Apart from that pure RNA was

considered to have same values in the ratios A260A23o and A260A18o (lmbeaud et ai 2005)

But the checking of purity of RNA using UV spectrophotometer was not really sensitive as

it is easily affected by presence of contaminants (Strand et ai 2007) Over estimation of

concentration ofRNA caused by the absorbance at 260run indicated the presence ofgenomic

DNA (Imbeaud et ai 2005) While the absorbance at 280run signified the presence of

protein but not totally neglected the presence of other potential organic contaminants which

also interfered with the measurement (lmbeaud et ai 2005)

13

163 Considerations on Designing Primer

The amplification of genes of interest were accomplished by the activities of primers In

Older to observe the expression of specific gene in the sample primers were designed to

blrget the gene ofinterest The primer usuall y came in pair which are the forward and reverse

primers The primer pairs that were designed amplify intended target only According to Ye

et aI (2012) primer pairs which merely amplify the intended target were considered as

specific primer This is because the primer pairs have no any matches to other unintended

target in other words the primers were unable to amplify any undesired product by means

ofchanging its orientation or in any distances between the primer pairs (Ye et al 2012) In

the process of designing primers there were severa~ conditions that required to take into

consideration Ye et al (2012) stated that the characteristics of primer pairs includes small

difference in melting temperatures (Tm) of the primers a considerable GC content and

minimal homodimer heterodimer and hairpin structure Ye et al (2012) also pointed out

that a minimal base changes as few as possible or less than two base changes at the 3 end

allowed certain amplification efficiency Base mismatches in 5 end was more tolerable than

3 end base mismatches Ye et al (2012) stated that the primer have to be designed to flank

the region of interest

According to Gubelmann et al (2011) in the case ofthe gene comprised of transcript

variants the primers were designed to be either specific to the gene which is flanking the

common region of the variants or specific to the transcript which is covering one of the

splice variants The amplification of genomic DNA was prevented if the one of the primer

pair span the exon of the transcript (Gubelmann et al 2011) Moreover Ye et al (2012)

also stated that the amplification ofundesired genomic DNA able to be avoided with the one

primers span on one of the exon-exon junctions of the transcript variants

14

242 NPCIHKl cell line

There were a numbers of malignant nasopharyngeal cell lines found in China and Hong

Kong (Glaser et ai 1989) The NPC cell lines identified in Hong Kong was NPCHKl

(Huang et at 1980 Glaser et ai 1989) HKl cell line is a well-differentiated squamous

carcinoma which have the same morphology with the human tumour derived from male

Chinese patient (Huang et at 1980) Huang et ai (1980) confinned the HK 1 as disease cell

line through the tumours developed at the site of inoculation in athymic nude BALBc (nunu)

mice This is a type of mice missing thymus gland it has no T cells which facilitate the study

oftumour

25 Analysis of Gene Expression

Barber Hanner Coleman amp Clark (2005) stated that analysis of gene expression IS

conducted in the situation that genes from different types of tissue need to be compared In

this study differential expression of RPS15 gene is compared between nonnal and tumour

cells

251 Housekeeping gene as a control

The differential gene expression is detected by the using the techniques ofpolymerase chain

reaction (PCR) The quantity of the RNA used in batch-to-batch variations in PCR reagents

need to be controlled (Barber et ai 2005) Barber et at (2005) stated that to nonnalize the

gene expression data housekeeping gene is used as a control In this study glyceraldehydeshy

3-phosphate dehydrogenase is utilized as a control Kozera and Rapacz (2013) mentioned

that a housekeeping gene have to conserve its expression level

11

1511 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene

GAPDH gene is assigned as a control because it controls for a nonnal glycolysis pathway in

normal cell lines The GAPDH gene found in tumour cell lines have an uncontrolled

glycolysis and cell growth GAPDH gene is used as a nonnal indicator in the gene expression

analysis

16 Principles of Methodology

161 The Advantages of TRIzol Reagent

The total RNA extracted using TRlzol reagent is not contaminated with DNA and protein

(Ma Young amp SheD 2008) Ma et al (2008) stated the process of total RNA isolation also

achievable within an hour Ahmann et al (2008) mentioned that the use of TRlzol reagent

capable in obtaining variety of genetic material for example RNA DNA and protein

Besides TRlzol reagent have a powerful benefits in which it is able to process large

amount of sample where a lot of high quality RNA is obtained using a large amount of

plasma cells (Ahmann et al 2008)

12

262 Qualitative and Quantitative Analysis of RNA

The commonly used method to determine the integrity of total RNA extracted is AGE The

band intensities of ribosomal bands indicates the integrity of the total RNA A completely

intact RNA was observed to have two distinct and clear bands which corresponded to 28S

rRNA band and 18S rRNA band The extracted RNA that shown its 28S ribosomal band

intensity doubled that of 18S ribosomal band is considered intact RNA ofhigh quality where

the ratio of 28S to 18S was approximately 2 1 (Imbeaud et al 2005) According to

Wieczorek Delauriere and Schagat (2012) the functions of AGE not only determine the

integrity ofthe RNA it also able to detect the degradation ofRNA Strand Enell Hedenfalk

and Ferno (2007) mentioned that analysis of gene expression was influenced by the quality

of RNA specifically the degradation in its quality The concentration of RNA was usually

quantified by using UV-spectrophotometer (UV-spec) During the quantification process

absorbance at various wavelength were detected According to Imbeaud et al (2005) RNA

which is truly pure was determined to record a value of more than or equal to 18 in the

absorbance ratio at wavelength 260run to 280run (A160A28IJ) Apart from that pure RNA was

considered to have same values in the ratios A260A23o and A260A18o (lmbeaud et ai 2005)

But the checking of purity of RNA using UV spectrophotometer was not really sensitive as

it is easily affected by presence of contaminants (Strand et ai 2007) Over estimation of

concentration ofRNA caused by the absorbance at 260run indicated the presence ofgenomic

DNA (Imbeaud et ai 2005) While the absorbance at 280run signified the presence of

protein but not totally neglected the presence of other potential organic contaminants which

also interfered with the measurement (lmbeaud et ai 2005)

13

163 Considerations on Designing Primer

The amplification of genes of interest were accomplished by the activities of primers In

Older to observe the expression of specific gene in the sample primers were designed to

blrget the gene ofinterest The primer usuall y came in pair which are the forward and reverse

primers The primer pairs that were designed amplify intended target only According to Ye

et aI (2012) primer pairs which merely amplify the intended target were considered as

specific primer This is because the primer pairs have no any matches to other unintended

target in other words the primers were unable to amplify any undesired product by means

ofchanging its orientation or in any distances between the primer pairs (Ye et al 2012) In

the process of designing primers there were severa~ conditions that required to take into

consideration Ye et al (2012) stated that the characteristics of primer pairs includes small

difference in melting temperatures (Tm) of the primers a considerable GC content and

minimal homodimer heterodimer and hairpin structure Ye et al (2012) also pointed out

that a minimal base changes as few as possible or less than two base changes at the 3 end

allowed certain amplification efficiency Base mismatches in 5 end was more tolerable than

3 end base mismatches Ye et al (2012) stated that the primer have to be designed to flank

the region of interest

According to Gubelmann et al (2011) in the case ofthe gene comprised of transcript

variants the primers were designed to be either specific to the gene which is flanking the

common region of the variants or specific to the transcript which is covering one of the

splice variants The amplification of genomic DNA was prevented if the one of the primer

pair span the exon of the transcript (Gubelmann et al 2011) Moreover Ye et al (2012)

also stated that the amplification ofundesired genomic DNA able to be avoided with the one

primers span on one of the exon-exon junctions of the transcript variants

14

1511 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene

GAPDH gene is assigned as a control because it controls for a nonnal glycolysis pathway in

normal cell lines The GAPDH gene found in tumour cell lines have an uncontrolled

glycolysis and cell growth GAPDH gene is used as a nonnal indicator in the gene expression

analysis

16 Principles of Methodology

161 The Advantages of TRIzol Reagent

The total RNA extracted using TRlzol reagent is not contaminated with DNA and protein

(Ma Young amp SheD 2008) Ma et al (2008) stated the process of total RNA isolation also

achievable within an hour Ahmann et al (2008) mentioned that the use of TRlzol reagent

capable in obtaining variety of genetic material for example RNA DNA and protein

Besides TRlzol reagent have a powerful benefits in which it is able to process large

amount of sample where a lot of high quality RNA is obtained using a large amount of

plasma cells (Ahmann et al 2008)

12

262 Qualitative and Quantitative Analysis of RNA

The commonly used method to determine the integrity of total RNA extracted is AGE The

band intensities of ribosomal bands indicates the integrity of the total RNA A completely

intact RNA was observed to have two distinct and clear bands which corresponded to 28S

rRNA band and 18S rRNA band The extracted RNA that shown its 28S ribosomal band

intensity doubled that of 18S ribosomal band is considered intact RNA ofhigh quality where

the ratio of 28S to 18S was approximately 2 1 (Imbeaud et al 2005) According to

Wieczorek Delauriere and Schagat (2012) the functions of AGE not only determine the

integrity ofthe RNA it also able to detect the degradation ofRNA Strand Enell Hedenfalk

and Ferno (2007) mentioned that analysis of gene expression was influenced by the quality

of RNA specifically the degradation in its quality The concentration of RNA was usually

quantified by using UV-spectrophotometer (UV-spec) During the quantification process

absorbance at various wavelength were detected According to Imbeaud et al (2005) RNA

which is truly pure was determined to record a value of more than or equal to 18 in the

absorbance ratio at wavelength 260run to 280run (A160A28IJ) Apart from that pure RNA was

considered to have same values in the ratios A260A23o and A260A18o (lmbeaud et ai 2005)

But the checking of purity of RNA using UV spectrophotometer was not really sensitive as

it is easily affected by presence of contaminants (Strand et ai 2007) Over estimation of

concentration ofRNA caused by the absorbance at 260run indicated the presence ofgenomic

DNA (Imbeaud et ai 2005) While the absorbance at 280run signified the presence of

protein but not totally neglected the presence of other potential organic contaminants which

also interfered with the measurement (lmbeaud et ai 2005)

13

163 Considerations on Designing Primer

The amplification of genes of interest were accomplished by the activities of primers In

Older to observe the expression of specific gene in the sample primers were designed to

blrget the gene ofinterest The primer usuall y came in pair which are the forward and reverse

primers The primer pairs that were designed amplify intended target only According to Ye

et aI (2012) primer pairs which merely amplify the intended target were considered as

specific primer This is because the primer pairs have no any matches to other unintended

target in other words the primers were unable to amplify any undesired product by means

ofchanging its orientation or in any distances between the primer pairs (Ye et al 2012) In

the process of designing primers there were severa~ conditions that required to take into

consideration Ye et al (2012) stated that the characteristics of primer pairs includes small

difference in melting temperatures (Tm) of the primers a considerable GC content and

minimal homodimer heterodimer and hairpin structure Ye et al (2012) also pointed out

that a minimal base changes as few as possible or less than two base changes at the 3 end

allowed certain amplification efficiency Base mismatches in 5 end was more tolerable than

3 end base mismatches Ye et al (2012) stated that the primer have to be designed to flank

the region of interest

According to Gubelmann et al (2011) in the case ofthe gene comprised of transcript

variants the primers were designed to be either specific to the gene which is flanking the

common region of the variants or specific to the transcript which is covering one of the

splice variants The amplification of genomic DNA was prevented if the one of the primer

pair span the exon of the transcript (Gubelmann et al 2011) Moreover Ye et al (2012)

also stated that the amplification ofundesired genomic DNA able to be avoided with the one

primers span on one of the exon-exon junctions of the transcript variants

14

262 Qualitative and Quantitative Analysis of RNA

The commonly used method to determine the integrity of total RNA extracted is AGE The

band intensities of ribosomal bands indicates the integrity of the total RNA A completely

intact RNA was observed to have two distinct and clear bands which corresponded to 28S

rRNA band and 18S rRNA band The extracted RNA that shown its 28S ribosomal band

intensity doubled that of 18S ribosomal band is considered intact RNA ofhigh quality where

the ratio of 28S to 18S was approximately 2 1 (Imbeaud et al 2005) According to

Wieczorek Delauriere and Schagat (2012) the functions of AGE not only determine the

integrity ofthe RNA it also able to detect the degradation ofRNA Strand Enell Hedenfalk

and Ferno (2007) mentioned that analysis of gene expression was influenced by the quality

of RNA specifically the degradation in its quality The concentration of RNA was usually

quantified by using UV-spectrophotometer (UV-spec) During the quantification process

absorbance at various wavelength were detected According to Imbeaud et al (2005) RNA

which is truly pure was determined to record a value of more than or equal to 18 in the

absorbance ratio at wavelength 260run to 280run (A160A28IJ) Apart from that pure RNA was

considered to have same values in the ratios A260A23o and A260A18o (lmbeaud et ai 2005)

But the checking of purity of RNA using UV spectrophotometer was not really sensitive as

it is easily affected by presence of contaminants (Strand et ai 2007) Over estimation of

concentration ofRNA caused by the absorbance at 260run indicated the presence ofgenomic

DNA (Imbeaud et ai 2005) While the absorbance at 280run signified the presence of

protein but not totally neglected the presence of other potential organic contaminants which

also interfered with the measurement (lmbeaud et ai 2005)

13

163 Considerations on Designing Primer

The amplification of genes of interest were accomplished by the activities of primers In

Older to observe the expression of specific gene in the sample primers were designed to

blrget the gene ofinterest The primer usuall y came in pair which are the forward and reverse

primers The primer pairs that were designed amplify intended target only According to Ye

et aI (2012) primer pairs which merely amplify the intended target were considered as

specific primer This is because the primer pairs have no any matches to other unintended

target in other words the primers were unable to amplify any undesired product by means

ofchanging its orientation or in any distances between the primer pairs (Ye et al 2012) In

the process of designing primers there were severa~ conditions that required to take into

consideration Ye et al (2012) stated that the characteristics of primer pairs includes small

difference in melting temperatures (Tm) of the primers a considerable GC content and

minimal homodimer heterodimer and hairpin structure Ye et al (2012) also pointed out

that a minimal base changes as few as possible or less than two base changes at the 3 end

allowed certain amplification efficiency Base mismatches in 5 end was more tolerable than

3 end base mismatches Ye et al (2012) stated that the primer have to be designed to flank

the region of interest

According to Gubelmann et al (2011) in the case ofthe gene comprised of transcript

variants the primers were designed to be either specific to the gene which is flanking the

common region of the variants or specific to the transcript which is covering one of the

splice variants The amplification of genomic DNA was prevented if the one of the primer

pair span the exon of the transcript (Gubelmann et al 2011) Moreover Ye et al (2012)

also stated that the amplification ofundesired genomic DNA able to be avoided with the one

primers span on one of the exon-exon junctions of the transcript variants

14

163 Considerations on Designing Primer

The amplification of genes of interest were accomplished by the activities of primers In

Older to observe the expression of specific gene in the sample primers were designed to

blrget the gene ofinterest The primer usuall y came in pair which are the forward and reverse

primers The primer pairs that were designed amplify intended target only According to Ye

et aI (2012) primer pairs which merely amplify the intended target were considered as

specific primer This is because the primer pairs have no any matches to other unintended

target in other words the primers were unable to amplify any undesired product by means

ofchanging its orientation or in any distances between the primer pairs (Ye et al 2012) In

the process of designing primers there were severa~ conditions that required to take into

consideration Ye et al (2012) stated that the characteristics of primer pairs includes small

difference in melting temperatures (Tm) of the primers a considerable GC content and

minimal homodimer heterodimer and hairpin structure Ye et al (2012) also pointed out

that a minimal base changes as few as possible or less than two base changes at the 3 end

allowed certain amplification efficiency Base mismatches in 5 end was more tolerable than

3 end base mismatches Ye et al (2012) stated that the primer have to be designed to flank

the region of interest

According to Gubelmann et al (2011) in the case ofthe gene comprised of transcript

variants the primers were designed to be either specific to the gene which is flanking the

common region of the variants or specific to the transcript which is covering one of the

splice variants The amplification of genomic DNA was prevented if the one of the primer

pair span the exon of the transcript (Gubelmann et al 2011) Moreover Ye et al (2012)

also stated that the amplification ofundesired genomic DNA able to be avoided with the one

primers span on one of the exon-exon junctions of the transcript variants

14