Shunxin Zheng. Cell Communications 2017 June 2017

19

Screening for interacting protein of senescence marker protein 30 in SK-Hep-1 Hepatoma cell

Shunxin Zheng1, Zhijing Mo1, Kun Li1, Zhilue Lv1, Yuan Zhuang1, Meiyu

Xie, Shunli Guo, Sufang Zhou1* 1 Department of Biochemistry and Molocular Biology, School of Pre-

clinical Medicine, Guangxi Medical University, Nanning, Guangxi

530021, China

Abstract Objective: Senescence marker protein 30 (SMP30) is a hepatocellular carcinoma (HCC)

associated antigen，and previous experiments have confirmed that it can inhibit the

proliferation, migration, apoptosis and cycle of HCC cells. However, whether these effects

are related to SMP30 interacting proteins is unclear. Methods: Flag-SMP30 lentiviral

vector was constructed and transfected into hepatoma cell line SK-Hep-1, and the over

expression and empty stable cell lines were constructed. RT-PCR and Western blotting

were used to detect the expression of SMP30. Total cellular protein was extracted for co-

immunoprecipitation (CO-IP), and the eluted proteins were identified by mass

spectrometry analysis. Results: SMP30 protein was highly expressed in transfected

hepatoma cell lines, and the Mass Spectrometry results showed that there were 266 direct

or indirect interaction proteins. These proteins are closely related to bacterial infection,

tumor development, DNA replication, cytoskeleton regulation, RNA transport, glucose

metabolism and other biological processes. Conclusion: In this study, we have screened

out the proteins interacting with SMP30, which laid the foundation for the next step of

protein interaction verification and studying the mechanism of SMP30 in HCC.

Key words: SMP30, Interactive protein, SK-Hep-1, CO-IP, Mass spectrum

†These authors contributed equally to the work

* Corresponding Author: Department of Biochemistry and Molecular Biology,

School of Pre-clinical Medicine, Guangxi Medical University, Nanning,

Guangxi 530021, China. Email:zsf200000@163.com

Cell Communications

Received: 13 February, 2017 Published: 01 June, 2017

Shunxin Zheng. Cell Communications 2017 June 2017

20

Introduction

SMP30 is a calcium-binding protein, which is

mainly present in the liver and kidney tissue

cells, especially its content is more abundant in

the liver cell [1, 2, 3]. By SEREX experimental

technique, SMP30 has been identified as one of

the HCC associated antigens [4]. The

expression of SMP30 in HCC and adjacent

tissues was significantly different, its

expression in HCC is low and high in the

adjacent tissues. This differential expression

may be related to its biological role in the

occurrence and development of HCC [5].

Immunohistochemical analysis showed that the

expression of SMP30 was closely related to the

survival and prognosis of HCC patients [6].

Anti-SMP30 autoantibody is found in sera of

patients with HCC, which can be used as a

serological marker for the diagnosis of HCC [7,

8]. The results of cell characterization showed

that the proliferation, migration, invasion,

apoptosis and cell cycle were up-regulated after

siRNA inhibited the expression of SMP30 in

HepG2 cells [9]. In conclusion, SMP30 plays

an important role in the development and

progression of HCC. However, in the process

of HCC, the interaction protein of SMP30 and

its mechanism need to be further studied. In this

study, we used co-immunoprecipitation-

tandem mass spectrometry technology to

analyze the interaction protein of SMP30 in

hepatoma cells.

Materials and methods

SK-Hep-1 hepatoma cell line was purchased

from Kunming cell bank. Magnetic beads and

Flag-SMP30 lentivirus packaging services

from Guangzhou Huijun biotechnology

company. Anti-SMP30 monoclonal antibody

was obtained from Abcam . Anti-Flag

monoclonal antibody was purchased from

Sigma-Aidrich . All other reagents were

analytical grade.

Methods

1. Flag-SMP30 lentivirus transfected SK-Hep-

1 hepatoma cells.

SK-Hep-1 cells (5 × 106cells/well) were

inoculated into 6-well plates and 3 wells were

inoculated. After the cells were adhered to the

plate 40% -50%, each well was replaced with 1

ml of DMEM complete medium containing 10

µg/ml polybrene. Placed in 37℃ incubation for

1h. One ml of the lentivirus of Flag-SMP30

was added to the first well, 1 ml of the

lentivirus of empty vector was added to the

second well, and the third well was used as

blank control. After shaking, the cells were

incubated for 6 hours. The medium in the wells

was removed, washed twice with PBS, and

replaced with DMEM complete medium.

When the cells grew to 80% confluences, 1 μL

of puromycin (2 mg/mL) was added to the first

and second wells for drug screening. The

medium was changed every day until all the

untransfected cells were apoptotic. RT-PCR

and Western blotting were used to detect the

expression of SMP30.

2. CO-

Immunoprecipitation (CO-IP)

Cells were scraped off with a spatula and

transferred to 1.5 ml Ep tubes, and then

centrifuged at 1000 rpm for 5 min. After cells

were collected and washed 3 times with PBS,

750 ul of precooled lysis solution was added

and mixed on dry ice for 10 min, and then lysed

at 4 °C for 30 min and centrifuged at 12000 rpm

for 10 min at 4 °C. After the centrifugation,

supernatant was transferred to a new Ep tube.

An appropriate amount of streptavidin beads,

washed 3 times with PBST, was added to the

supernatant obtained in the previous step, 4°C

overnight. The supernatant was discarded on a

magnetic separator. The beads were washed 3

times with cell lysis solution and washed once

with PBS, and then washed with biotin for 10

min. Elution was separated by magnetic

separator and pipetted into a new 1.5 ml Ep

tube.

Shunxin Zheng. Cell Communications 2017 June 2017

21

3. Mass spectrometry analysis The elution obtained from CO-IP was

precipitated with precooled acetone, and the

residual acetone in precipitation was blown dry.

The precipitation, adding appropriate amount

of cell lysis solution and 3x loading buffer, was

boiled for 5 minutes, cooled on ice and

separated by SDS-PAGE. The gel was stained

by Coomassie brilliant blue, the heavy and light

chains of the antibody were excised. All the

remaining bands were cut into 0.6 ml Ep tubes,

and identified by liquid phase tandem mass

spectrometry.

4. Mass spectrometry data analysis The interacting proteins obtained from the

mass spectrometry were subjected to GO

analysis to describe the molecular function,

Cellular Component and biological process.

Through KEGG pathway analysis could

determine the involvement of these proteins in

the biochemical metabolism and signal

transduction pathways. The STRING database

was used for enrichment analysis of known or

predicted interacting proteins.

Results

1. Expression of SMP30 in SK-Hep-1

Hepatoma Cells

SMP30 recombinant lentiviral vector and

empty lentiviral vector (LV5) were transfected

into SK-Hep-1 cell line respectively. The

expression levels of SMP30 in SK-Hep-1, SK-

Hep-1-SMP30 and SK-Hep-1-LV5 were

detected by RT-PCR (Figure.1A). The

expression of SMP30 in SK-Hep-1-SMP30 cell

line was significantly higher than that in the

other two cell lines (Figure.1B). Western

blotting was used to detect the expression of

SMP30 protein and Flag protein in three cell

lines (Figure.1C).

2. Gene ontology (GO) analysis and KEGG

pathway analysis of SMP30 interacting

proteins

The results of mass spectrometry analysis

showed that 266 proteins had direct or indirect

interaction with SMP30. These interacting

proteins were uploaded to the DAVID online

tool for Gene Ontology analysis. These

proteins are mainly involved in the cellular

components such as cell membrane, exosomes,

microtubules, vesicles and cytoplasm, and are

closely related to the biological processes of

protein catabolism, transcription complex

formation,

actin depolymerization and MAPK

signal pathway (Table1). KEGG pathway

analysis showed that these proteins were

closely related to bacterial infection, tumor

occurrence, DNA replication, cytoskeleton

regulation, RNA transport, glucose metabolism

and other biological processes (Table 2).

3. Prediction of SMP30 interacting protein

by STRING database

STRING (Search Tool for the Retrieval of

Interacting Genes/Proteins) is a database for

the study and collection of known or predicted

protein interactions. It uses the information of

neighborhood, fusion, co-occurrence,

homology, co-expression, experimental,

knowledge, text mining to study the direct or

indirect interaction proteins of target proteins.

There were 10 proteins interacted with RGN

protein through data screening (Figure 2).

These proteins are involved in glucose

metabolism (INS), protein synthesis (SEC16A),

apoptosis (PCNX), hemoglobin (HBA2),

histone acetylation (PHF16A) and other

biological processes. Unfortunately, these 10

proteins were not detected in this mass

spectrometric test. At the same time, we also

predicted the interaction of these proteins, and

the study found that their interaction protein

does not also exist in the results of mass

spectrometric analysis.

Shunxin Zheng. Cell Communications 2017 June 2017

22

Figure.1. ver-expressed and down-expressed SMP30 in SK-Hep-1 hepatoma cells. A. The

expression of SMP30 was detected by RT-PCR. B. The relative expression of SMP30. The

expression level of SK-Hep-1-LV5 was used as reference. C. The expression of SMP30 and Flag

protein was detected by Western blotting.

Table 1. GO analysis of interacting protein of SMP30

Count P Value

Biological Process formation of translation preinitiation complex(GO:0001731) 8 1.71E-08

regulation of translational initiation (GO:0006446) 8 1.49E-07

associated ubiquitin-dependent protein catabolic process (GO:0030433) 7 1.62E-04

proteolysis involved in cellular protein catabolic process (GO:0051603) 6 3.07E-04

negative regulation of p38MAPK cascade (GO:1903753) 3 1.73E-03

proteasome assembly (GO:0043248) 3 7.71E-03

actin polymerization or depolymerization (GO:0008154) 3 7.71E-03

Cellular Components

membrane( GO:0016020) 59 3.88E-22

extracellular exosome( GO:0070062) 92 5.83E-21

nuclear proteasome complex( GO:0031595) 7 4.43E-10

microtubule( GO:0005874) 14 1.30E-08

cytosolic proteasome complex( GO:0031597) 6 2.11E-08

nucleoplasm( GO:0005654) 47 9.87E-08

Cytoplasm( GO:0005737) 74 1.93E-07

focal adhesion( GO:0005925) 18 5.26E-06

COPI vesicle coat( GO:0030126) 5 1.97E-05

proteasome core complex( GO:0005839) 5 1.97E-05

Molecular Function

Shunxin Zheng. Cell Communications 2017 June 2017

23

poly(A) RNA binding(GO:0044822) 64 1.87E-11

proteasome-activating ATPase activity (GO:0036402) 6 9.64E-09

TBP-class protein binding (GO:0017025) 6 8.74E-08

ATP binding (GO:0005524) 46 1.87E-07

translation initiation factor activity (GO:0003743) 8 6.05E-06

enzyme regulator activity (GO:0030234) 4 5.93E-04

protein binding (GO:0005515) 4 3.63E-03

Table 2. KEGG pathway analysis of interacting protein of SMP30

KEGG PATHWAY Count P Value

Proteasome（cfa03050） 21 3.15E-21

Epstein-Barr virus infection（cfa05169） 22 1.50E-09

Biosynthesis of antibiotics（cfa01130） 15 5.05E-04

DNA replication（cfa03030） 6 1.47E-03

Pentose phosphate pathway（cfa00030） 5 3.51E-03

Biosynthesis of amino acids（cfa01230） 7 7.99E-03

Regulation of actin cytoskeleton（cfa04810） 12 1.11E-02

Carbon metabolism（cfa01200） 8 1.76E-02

Vasopressin-regulated water reabsorption（cfa04962） 5 1.84E-02

Aminoacyl-tRNA biosynthesis（cfa00970） 5 1.99E-02

NOD-like receptor signaling pathway（cfa04621） 5 3.03E-02

Salmonella infection（cfa05132） 6 3.39-02

RNA transport（cfa03013:） 9 3.62E-02

Fructose and mannose metabolism（cfa00051:） 4 3.63-02

Figure.2. Interaction of SMP30 and its interacting proteins

Shunxin Zheng. Cell Communications 2017 June 2017

24

Discussion

SMP30 is a hepatocellular carcinoma

associated antigen. Previous experiments have

proved that SMP30 can inhibit the proliferation,

migration, apoptosis and cycle of HepG2 cells

[9]. It is associated with the survival prognosis

of patients with HCC [6]. Searching for the

interaction protein of SMP30 in

hepatocarcinoma cells is the main content of

our study.

In this study, we transfected the Flag-

SMP30 gene into hepatocellular carcinoma cell

line SK-Hep-1 by lentiviral transfection，and

constructed a stable transfected cell line. After

extraction of total cellular protein, co-

immunoprecipitation experiments were carried

out using small-molecule antibodies against

Flag-tagged proteins 10, 11,12]. The anti - Flag

antibody has a lower molecular weight, which

can reduce the physical adhesion of nonspecific

interaction proteins and can effectively reduce

the false-positive protein 13, 14, 15 .

The results of mass spectrometry showed

that 266 proteins were directly or indirectly

interacted with SMP30 protein in SK-Hep-1

hepatoma cells. We used bioinformatics

software for gene function enrichment, 266

interacting proteins mainly participated in cell

membrane, cytoplasm, microtubules and

vesicles, as well as cell metabolism, protein

metabolism, signal transduction, actin

depolymerization and other biological

processes. These results suggest that SMP30

interacting proteins may regulate the

metabolism of cells through a variety of

signaling molecules, thus affect the occurrence

and development of HCC. KEGG signaling

pathway showed that 266 interacting proteins

were involved in viral infection, cell

metabolism, DNA replication and RNA

transport. The pathways of alcoholism and

HBV infection are potential factors in the

development of liver cancer [16, ,18].The

pathway of glucose metabolism and

cytoskeleton regulation is closely related to the

increase and metastasis of tumor [19, 20, 21].

At the same time, we also used the STRING

database to predict that 10 proteins could

theoretically interact with SMP30, but

unfortunately none of these 10 proteins were

detected in this mass spectrometry experiment.

The reason for the inconsistencies in the

STRING software prediction and mass

spectrometry analysis may be 1) The evidence

of the software prediction is mainly derived

from the existing experimental data and other

databases or the application of bioinformatics

methods such as chromosomal approach, gene

fusion and phylogenetic spectrum. There is a

significant difference between prediction

method and the actual situation. 2) The

research for SMP30 as hepatocellular

carcinoma-related antigen is less, the

experimental data are not sufficient 3)

Hepatoma cell selected in the experiment may

be subjected to individual differences, affecting

the results of the experiment.

In this study, the interaction proteins of

SMP30 in hepatocellular carcinoma cells were

analyzed by co-immunoprecipitation-tandem

mass spectrometry, but the results need to be

further confirmed by corresponding

experiments. The results of this experiment can

provide a new research direction and

theoretical basis for the follow-up study of

SMP30 protein.

Acknowledgments:

This work was supported by National Natural

Science Foundation of China (81572994,

81460432); Guangxi Natural Science

Foundation (2015GXNSFDA139017);

Guangxi Science and Technology Research

and Technology Development Project

(15104001-7); Guangxi Nanning Qingxiu

District Science and Technology Research and

[

[ ]

17

Shunxin Zheng. Cell Communications 2017 June 2017

25

Technology Development Project (2014S03).

References

1. Fujita T, Inoue H, Kitamura T, Sato N,

Shimosawa T, Maruyama N. Senescence

marker protein-30 (SMP30) rescues cell

death by enhancing plasma membrane

Ca(2+)-pumping activity in Hep G2

cells.Biochemical and biophysical

research communications. 1998. 250(2):

374-80.

2. Fujita T, Uchida K, Maruyama N.

Purification of senescence marker protein-

30 (SMP30) and its androgen-independent

decrease with age in the rat liver.

Biochimica et biophysica acta. 1992.

1116(2): 122-8.

3. Fujita T, Shirasawa T, Uchida K,

Maruyama N. Isolation of cDNA clone

encoding rat senescence marker protein-30

(SMP30) and its tissue distribution.

BiochimBiophysActa. 1992. 1132(3): 297-

305.

4. Stenner-Liewen F, Luo G, Sahin U, Tureci

O, Koslovski M, Kautz I, Liewen H,

Pfreundschuh M. Definition of tumor-

associated antigens in hepatocellular

carcinoma. Cancer epidemiology,

biomarkers & prevention. 2000. 9(3): 285-

90.

5. Zhou SF, Mo FR, Bin YH, Hou GQ, Xie

XX, Luo GR. Serum immunoreactivity of

SMP30 and its tissues expression in

hepatocellular carcinoma. Clinical

biochemistry. 2011. 44(4): 331-6.

6. Mo Z, Zheng S, Lv Z, Zhuang Y, Lan X,

Wang F, Lu X, Zhao Y, Zhou S.

Senescence marker protein 30 (SMP30)

serves as a potential prognostic indicator in

hepatocellular carcinoma. Scientific

reports. 2016. 6: 39376.

7. Zhou SF, Xie XX, Bin YH, Lan L, Chen F,

Luo GR. Identification of HCC-22-5

tumor-associated antigen and antibody

response in patients. Clinica chimica acta;

international journal of clinical chemistry.

2006. 366(1-2): 274-80.

8. Zhang SC, Huang P, Zhao YX, Liu SY, He

SJ, Xie XX, Luo GR, Zhou SF. Soluble

expression of recombinant human SMP30

for detecting serum SMP30 antibody

levels in hepatocellular carcinoma patients.

Asian Pacific journal of cancer prevention.

2013. 14(4): 2383-6.

9. Zhang SC, Liang MK, Huang GL, Jiang K,

Zhou SF, Zhao S. Inhibition of SMP30

gene expression influences the biological

characteristics of human HepG2 cells.

Asian Pacific journal of cancer prevention.

2014. 15(3): 1193-6.

10. Paul FE, Hosp F, Selbach M. Analyzing

protein-protein interactions by quantitative

massspectrometry. Methods. 2011. 54(4):

387-95.

11. Bain AL, Harris JL, Khanna KK.

Identification of ATM-Interacting Proteins

by Co-immunoprecipitation and

Glutathione-S-Transferase (GST) Pull-

Down Assays. Methods in molecular

biology. 2017. 1599: 163-181.

12. Han J, Zhang M, Froese S, Dai FF,

Robitaille M, Bhattacharjee A, Huang X,

Jia W, Angers S, Wheeler MB, Wei L. The

Identification of Novel Protein-Protein

Interactions in Liver that Affect Glucagon

Receptor Activity. PLoS One. 2015. 10(6):

e0129226.

13. Zilio N, Boddy MN. Improved Tandem

Affinity Purification Tag and Methods for

Isolation of Proteins and Protein

Complexes from Schizosaccharomyces

pombe. Cold Spring Harb Protoc. 2017.

2017(3): pdb.prot091611.

14. Gerace E, Moazed D. Affinity Pull-Down

of Proteins Using Anti-FLAG M2 Agarose

Beads. Methods in enzymology. 2015. 559:

99-110.

15. Babu M, Kagan O, Guo H, Greenblatt J,

Emili A. Identification of protein

Shunxin Zheng. Cell Communications 2017 June 2017

26

complexes in Escherichia coli using

sequential peptide affinity purification in

combination with tandem mass

spectrometry. Journal of visualized

experiments. 2012. (69).

16.

Suh B, Yun JM, Park S, Shin DW, Lee TH,

Yang HK, Ahn E, Lee H, Park JH, Cho B.

Prediction of future hepatocellular

carcinoma incidence in moderate to heavy

alcohol drinkers with the FIB-4 liver

fibrosis index. Cancer. 2015. 121(21):

3818-25.

17.

Sherman M, Shafritz DA. Hepatitis B virus

and hepatocellular carcinoma: molecular

biology and mechanistic considerations.

Seminars in liver disease. 1984. 4(2): 98-

112.

18.

Qin Y, Zhong Y, Ma T, Wu F, Wu H, Yu H,

Huang C, Li Z. Alteration of liver

glycopatterns during cirrhosis and tumor

progression induced by HBV.

Glycoconjugate journal. 2016. 33(2): 125-

36.

19.

Berger E, Vega N, Weiss-Gayet M, Géloën

A. Gene Network Analysis of Glucose

Linked Signaling Pathways and Their Role

in Human Hepatocellular Carcinoma Cell

Growth and Survival in HuH7 and HepG2

Cell Lines. BioMed research international.

2015. 2015: 821761.

20.

Hu J, Niu M, Li X, Lu D, Cui J, Xu W, Li

G, Zhan J, Zhang H. FERM domain-

containing protein FRMD5 regulates cell

motility via binding to integrin β5 subunit

and ROCK1. FEBS Letters. 2014. 588(23):

4348-56.

21.

Liu F, Zhang W, You X, Liu Y, Li Y, Wang

Z, Wang Y, Zhang X, Ye L. The

oncoprotein HBXIP promotes glucose

metabolism reprogramming via

downregulating SCO2 and PDHA1 in

breast cancer. Oncotarget. 2015. 6(29):

27199-213.