The Modeling And Analysis Of Gene Regulatory Networks In HBV-related Hepatocellular Carcinoma

Primary hepatocellular carcinoma caused by chronic hepatitis B virus(HBV)infection depends on a variety of genes,transcriptomes,protein and their interactions and regulation.From the perspective of a single gene,the alteration of its expression profiles only explains the local occurrence of liver cancer but not in the overall and comprehensive understanding.The biological networks,a simulation and reconstruction of the regulatary relationship in genes based on the microarray data,is essential to visually demonstrate the relationship among the various functional elements in each biological system.In our study,the WGCNA was applied to simulate complex regulatory networks in order to find out the hub genes during the development of the HBV-related hepatocellular carcinoma.Subsequently,we tried to verify the gene prediction reliability of these key nodes by the following biochemical experiments.Our study focused on the screening of these hub genes and the investigation of their roles in the HepG2 cell line,an in vitro cellular model of the HBV-related HCC as an evidence whether the bioinformatics tool is helpful to search for the potential targets during the incidence and the development of the HBV-related HCC.The data were collected from 61 cancer samples and 63 para-carcinoma healthy tissues on the Agilent gene chips,on which the genes of HBV-related HCC were differently expressed.5968 different genes was summarized to establish a co-expressed scale-free network model by the WGCNA.Among them,13 co-expression modules by the clustering analysis were obtained.Their association with HCC were tested and ultimately visually displayed.Combined with the enrichment analysis and the literature survey,we finally found 13 hub genes in co-expression modules that closely related to GO classification,including epithelial cell differentiation,extracellular matrix remodeling,cell cycle etc.Further experiments were performed to verify the biological functions of one of the selected hub genes,given consideration to the comparative analysis and literature survey,the SHANK-associated RH domain interacting protein(SHARPIN).In vitro experiment,the eukaryotic over-expression plasmid pc DNA3.1(+)-SHARPIN and interferential plasmid pSUPER-shSHARPINs were respectively transfected into the human hepatoma cell line HepG2 to investigate the role of SHARPIN on the proliferation,the migration and the apoptosis.The results showed that SHARPIN over-expression mainly modulated the proliferation and the migration of HepG2 cells.The cell proliferation and migration were inhibited during the intracellular SHARPIN expression profile was interfered,while no significant effect was observed on the cell apoptosis.In summary,this study analyzed data sets of the HBV-related HCC through a bioinformatic protocol,the WGCNA,constructing a set of gene interaction network.The obtained 13 co-expression modules were used to identify the hub genes associated with the occurence and the development of HCC.Given consideration to the functionalanalysis and the literature surveys,we found that most of the hub genes were mentioned in the previous relevant literature,from which,for example,the SHARPIN was reported involved in the NF-?B pathway is believed to be closely related to the occurrence and development of human prostate cancer.In our study,the RNA interference was applied to verify the biological function of SHARPIN gene in hepatoma cells.It has confirmed that WGCNA may be an helpful method to identify and to establish biologically significantly gene modules.The understanding of the role of the selected hub genes on the signaling pathways and regulation networks would be of significance to discover novel diagnostic biomarkers and drug targets during the chronic HBV leading HCC.