Integrative network based analysis of mRNA and microRNA expression

Integrative network based analysis of mRNA and microRNA expression in vitamin D3-treated cancer cells

Susan Steinbusch-Coort, PhD

Dept. Bioinformatics-BiGCaT, NUTRIM, Maastricht University [email protected]

The Science of Big-Data Analytics & Visualization 23 November 2015 2

Nutritional Systems Biology

● Understanding nutritional processes at a systems level ● Integrating the effects of nutritional compounds at the gene

expression level with information on the regulatory level.

https://wellnessfx.files.wordpress.com/2011/06/picture-11.png


Nutrigenomics

http://www.alive.com/articles/view/23381/the_nutrigenomics_frontier


Nutrition and Epigenetics

www.int.laborundmore.de

http://www.int.laborundmore.de/archive/937688/Cancer-prevention-and-nutrition.html


Vitamin D3 metabolism

Deeb, KK, et al. "Vitamin D signalling pathways in cancer: potential for anticancer therapeutics." Nature Reviews Cancer (2007)


Vitamin D3-mediated regulation

Deeb, KK, et al. "Vitamin D signalling pathways in cancer: potential for anticancer therapeutics." Nature Reviews Cancer (2007)


Goal of this study

Pathway and network-based methods

Integrate mRNA and microRNA

expression data

Investigate regulatory action of

vitamin D3 in prostate cancer


Workflow


Multi-omics dataset

Human prostate cancer cell line

LNCaP - Lymph node metastasis in Caucasian male

100 nM 1,25 dihydroxyvitamin D3

control group

(n=4)

VitD-treated group

(n=4)

Transcriptomics Nimblegen-HG18-4plex

whole genome microarrays

GEO: GSE17461

MicroRNA-omics Agilent Human microRNA v3

microarrays

GEO: GSE23814

RNA isolation

48h treatment


Gene-level statistics

Quality Control and statistical analysis o performed by Wang et al. o one way ANOVA (p-value < 0.05) o correction for multiple testing

Transcriptomics data o fold change > 1.5 o p-value < 0.05

MicroRNA data o fold change > 2 o p-value < 0.05

Up-regulated

Down-regulated

420 413

Up-regulated

Down-regulated

9 0


VDR targets

CYP24A1 - degradation of vitamin D3 ↑

ORM1/ORM2 - acute phase plasma

protein ↑

CDKN2D/2C - cell growth regulator ↓

Literature study ● 25 publications and books ● 178 human VDR targets ● 21 changed genes


Workflow


Pathway analysis

PathVisio o Open source pathway analysis toolbox

o Data visualization and over-representation analysis

o www.pathvisio.org

WikiPathways o Collaborative pathway database

o 276 pathways in curated collection

o www.wikipathways.org

Kutmon, M, et al. "WikiPathways: capturing the full diversity of pathway knowledge." Nucleic Acids Res (2015)

Kutmon, M, et al. "PathVisio 3: An Extendable Pathway Analysis Toolbox." PLoS Comput Biol. (2015)

www.pathvisio.org

www.wikipathways.org


Pathway analysis

Transcriptomics

dataset

Pathway database

WikiPathways

Differentially expressed genes

Calculates Z-Score for each pathway

Over-representation analysis

Ranked list of pathways

Data visualization on pathway

diagrams


Pathway analysis

● Significantly altered pathways:

o 8 general cell cycle related pathways

o 7 cancer related pathways

Pathway Z-Score Category

DNA Replication 11.91 general

Cell Cycle 11.04 general

Histone Modifications 10.44 general

G1 to S cell cycle control 9.12 general

DNA damage response 5.40 general

ATM Signaling pathway 4.87 general

Fluoropyrimidine Activity 4.16 general

AhR signaling pathway 2.47 general

Pathway Z-Score Category

Retinoblastoma (RB) in Cancer 12.63 cancer

Gastric cancer network 1 10.44 cancer

Gastric cancer network 2 5.13 cancer

Integrated Pancreatic Cancer

Pathway

4.08 cancer

Integrated Cancer pathway 3.85 cancer

Integrated Breast Cancer Pathway 3.41 cancer

Signaling Pathways in

Glioblastoma

2.02 cancer


Pathway analysis

● Most of the pathways are down-regulated after vitamin D treatment

Cell Cycle Pathway Gastric Cancer Network 1

down

up


Workflow


Network building

Cytoscape o Network visualization and analysis tool

o Extendable through apps

o www.cytoscape.org

WikiPathways App WikiPathways web

service client and

GPML file format

importer

Shannon, P et al. "Cytoscape: a software environment for integrated models of biomolecular interaction networks." Genome research (2003)

www.cytoscape.org


Network building

Central genes:

TP53, CDKN1A and CDK2

linking 5 out of 8 pathways

WikiPathways App


Network building

Kutmon, M, et al. "WikiPathways App for Cytoscape: making biological pathways amenable to network analysis and visualization." F1000Research (2014)

up

down

VDR

WikiPathways App


Workflow


Network extension

● Problem: Only ~50% of protein coding genes are in pathways

changed genes in complete dataset

changed genes in all pathways

changed genes in altered general

pathways

833 420 up + 413 down

390 205 up + 185

down

73 14 up + 59 down

What about all the differentially expressed genes that are not in

the altered pathways?


Network extension ● Identify known protein-protein interaction

partners of the genes in the selected pathways

Database First neighbours

STRING database http://string-db.org/

443 changed genes

Database First neighbours

ENCODE http://encodenets.gersteinlab.org/

67 changed genes

● Identify known transcription factor-target interactions of

the genes in the selected pathways

support from Georg

Summer

http://string-db.org/



http://encodenets.gersteinlab.org/


VitD-extended network

● 583 out of 833 changed genes (~70%)

● 238 up ● 345 down

up-regulated

down-regulated


Workflow


Active network modules

jActiveModules App Finds clusters where member nodes show significant changes in

expression levels

● Connected sub-networks that are regulated by

Vitamin D treatment ● jActiveModules finds multiple active networks

with different scores o robust highest scoring down-regulated

module o 193 nodes o 41 from altered pathways


Active network modules

jActiveModules App

Module in vitD-extended network


Active network modules jActiveModules App

● 193 genes

o 192 DE genes, all down-regulated o 1 gene not DE (E2F4)

● 41 genes from pathway network o 22 in more than one pathway

What is the function of all genes in this active sub-network?


Functional enrichment

● Find GO processes in which the 193 genes of the active sub-network are over-represented

● ClueGO creates network of related GO terms

ClueGO App



Workflow


Vit D3-microRNA network CyTargetLinker App

Extends biological networks

with regulatory interactions

● TargetScan + miRTarBase

o 1,439 miRNAs → 25,886 miRNA-target

interactions

● 6 out of 9 changed miRNAs present in vitD-microRNA network

● Extend biological network with regulatory information o microRNAs, transcription factors, drugs, ...


Vit D3-microRNA network

31 targets up-regulated (3 in pathways) 23 targets down-regulated (4 in pathways)

Targeted by multiple microRNAs:

CLSPN - cell cycle

FZD5 - receptor for Wnt proteins

CACNG4 - calcium channel

CyTargetLinker App


Summary

● Data integration o Multi-omics datasets o Pathway and interaction resources

● Several cell cycle related and cancer-related pathways are down-regulated after vitamin D treatment

● Possible regulatory mechanism of vitamin D through microRNAs


Summary

● Combination of the network-based tools PathVisio and Cytoscape

● Straightforward, in-depth and biological meaningful analysis

● Integration of different multi-omics data in a network-based approach


Acknowledgments

Department of Bioinformatics, Maastricht University Martina Summer-Kutmon Kim de Nooijer Claire Lemmens Chris Evelo Lars Eijssen, Egon Willighagen, Linda Rieswijk, Frederieke Ehrhart, Anwesha Bohler-Dutta, Elisa Cirillo, Nuno Nunes, Jonathan Mélius, Ryan Miller. Department of Cardiology, Maastricht University Georg Summer

Dataset: Wei-Lin Wang et al.


Questions

Data & Analytics

Integrative network based analysis of mRNA and microRNA expression