Hepatitisc Virus Core Protein Epigenetic Silencing Of Wntantagonist SFRP1and Its Molecular Pathogenic Mechanisms

Objective: Hepatocellular carcinoma is the fifth most commonmalignant tumor wordwide.In recent years, its incidence and mortality rateare on the rise. Hepatitis C Virus infection is a major cause of thehepatocellular carcinoma, but the molecular events that lead tohepatocarcinogenesis during HCV infection are poorly understood. HCVcore protein encoded by Hepatitis C Virus is a multifunctional protein.Ithas been shown to interact with some improtant intracellular signalingpathways and transcription factors, to induce dysregulation of oncogenesand tumor suppressor genes,which result in the promotion of cellproliferation and cell malignant transformation,thus interfering with hostimmunogical defense, contributing to viral persistence, and eventuallyinvolving in HCC pathogenesis.Aberrant activation of Wnt/β-catenin signaling pathway contributes tothe hepatocellular carcinogenesis.Activation of β-catenin is found in50-83%of hepatocellular carcinomas,though CTNNB1(β-catenin)mutations are seen in only about20%of HCCs,and APC mutation is not seen in HCC.These results suggest that mechanisms other than CTNNB1mutation such as wnt antagonists SFRP (Secreted frizzled-related protein)genes may contribute to the activation of Wnt signaling in livercarcinognesis.DNA methylation is the improtant way of epigenetic modification.Rec-ent studies have also identified DNA hypermethylation in SFRP genes in avariety of human tumours, including colon, oesophagus, stomach,bladdercancer and HCCs. These changes are typically associated with unscheduledsilencing of SFRPs genes.Frequent DNA hypermethylation and silencing ofSFRPs genes activates the Wnt signaling pathway in hepatocarcinogenesis.HCV core protein has been shown to induce activation of Wnt signalingpathway, however whether HCV core protein exerts any epigeneticregulation effects on SFRP genes has not been elucidated.In this study, weestablished HCV core-overexpression cell model,and examined whetherHCV core protein exerts effects on methylation of SFRPs promoter regionand silencing of SFRPs genes.Finally,we investigated the molecularmechanisms of epigenetic modification of SFRP1by HCV core protein.Methods: Real-time PCR and Western bolt analysis were conductedto screen down-regulated SFRPs genes in HCV core-overexpression cellmodel. And the restored expression level of SFRP1gene was determined inAdCore-infected cells treated with5′-Aza-2′-deoxycytidine(DAC). Theepigenetic effects of HCV core protein on SFRP1gene were assessed by Methylation specific PCR and Bisulfite DNA sequencing methods. Finally,the molecular mechanisms that HCV core protein epigenetic silencing SFR-Ps genes were further assessed by chromatin immunoprecipitation,immunoprecipitation analysis.Result：By usingAdenovirus system, we have successfully establishedand identified HCV core-overexpression cell model.And we also found thatthe expression level of SFRP1was apparently down-regulated in Adcore-infected Huh7,HepG2cells or Huh7-core stable cell line. As observed viaquantitative RT-PCR and western blotting anlysis,5′-Aza-2′-deoxycytidine(DAC) treatment effectively restored the expression level of SFRP1inHCV Adcore-infected Huh7cells, and the restord effects in core-overexpre-ssion cells dose-dependent on5′-Aza-2′-deoxycytidine.We furtherexamined the DNA methylation status on the SFRP1promoter region bymethylation specific PCR and bisulfite DNA sequencing. And themethylation analysis revealed that CpG dinucleotide islands closer totranscription start site in SFRP1promoters were significantlyhypermethylated in core-infected Huh7cells, indicating that HCV coreprotein can effecttively induce hypermethylation modification of SFRP1promoter region and thus resulting in expression silence of SFRP1gene.Chip assay was used to investigate the epigenetic molecular mechanisms ofSFRP1down-regulated by HCV core protein, we found that HCV coreprotein can increase the expression levels of DNA methyltransferase-1and histone deacetylase-1,recruit DNA methyltransferase-1and histonedeacetylase-1to SFRP1promoters, or leading to repress Acetyl-Histone H3to SFRP1promoters, resulting in epigenetic silencing of SFRP1gene.Conclusion: HCV core protein recruited DNMT1and HDAC1where-as repressed Acetyl-Histone H3to SFRP1promoters, and subsequentlysilenced SFRP1via DNA methylation of promoters and histonedeacetylation.