Epigenetic Silence Of Sfrps By Hepatitis B Virus X Protein Enhance Hepatoma Cell Tumorigenicity Through Aberrant Wnt Signaling Pathway

Objective: Hepatocellular carcinoma (HCC) is the third leading causeof cancer related death worldwide.One of the high risk factor associated withHCC is chronic hepatitis B viral (HBV) infection. At present all over theworld, about400million people are infected with HBV, China accounts forabout one-third. HBV is a partial double-stranded DNA virus with a3.2kbgenome that contains four open reading frames encoding viral DNApolymerase, viral envelope, core protein, and HBV X protein (HBx). HBxencoding154amino acid, plays a significant role in virus replication.Increasing evidence suggests that HBx play an important role in thedevelopment of HCC. As a multifunctional regulator, HBx is capable oftrans-activation or trans-repression of both viral and cellular genes throughits direct interaction with nuclear transcription factors. Further, HBx isinvolved in several oncogenic signal transduction pathways includingWnt/β-catenin, Ras/MAPK, SAPK/JNK, NF-κB, and FAK.The Wnt/β-catenin signaling pathway has been implicated in a widerange of biological processes including cell senescence, cell death, differentiation and metastasis. Previous studies have suggested that aberrantactivation of the Wnt/β-catenin signaling pathway may contributesignificantly to hepatocellular carcinogenesis. Except for genetic mutationof Wnt/β-catenin pathway components,8the epigenetic events can alsocontribute to abnormal activation of this signaling pathway in hepatomacells. Secreted frizzled-related proteins (SFRPs), a family of five secretedglycoproteins, are extracellular signaling molecules that directly bind Wntligands and antagonize Wnt signaling pathway. Downregulation bypromoter hypermethylation of the Wnt inhibitors, such as SFRPs has beenidentified in HBV-associated HCC. Conversely, restoration of Wntantagonists such as SFRP1or WIF1leads to reduction of tumor growth andangiogenesis in HCC through inhibition of Wnt/β-catenin signaling.Thesedata point to a critical role for epigenetic silencing of SFRPs genes inabnormal activation of Wnt/β-catenin signaling pathway and involved inHBV-associated hepatocarcinogenesis.Recent studies have found that HBx could promote cell proliferationand involve in the primary carcinoma by means of activating Wnt/β-cateninsignaling pathway, but it is unclear whether HBx plays a direct role inepigenetic regulation of SFRPs in HCC. This research focuses on a cellularlevel and tumor tissue specimens observation of HBx SFRPs epigeneticregulation and HBx lead to further research the molecular mechanism oftumorigenesis. Methods: we evaluated the expression of SFRP1and SFRP5inhuman HCCs Liver tissue samples were obtained from surgical specimenswith the informed consent of patients at the2nd Affiliated Hospital ofChongqing Medical University.35primary HCC samples and adjacentnon-tumorous liver tissues were extracted DNA, RNA, and proteins, thendetected the expression of SFRP1and SFRP5. Since SFRPs have beenshown to be transcriptionally inactivated in cancer cells through promotermethylation, we determined the methylation status of the SFRP1andSFRP5promoters by methylation-specific PCR(MSP) analysis. By directdetection of infection by HBx, SFRP1and SFRP5promoters activity wasmeasured by luciferase activity. To determine if HBx support cell growth, weexamined the effects of Dnmt1silencing or SFRP1, SFRP5restoration onthe growth of HCC cells.Results: We collected35HCC tissues at the2nd Affiliated Hospital ofChongqing Medical University.We found that SFRP1and SFRP5transcripts were significantly decreased in HCCs relative to pairednon--tumorous liver tissues, which was further confirmed by western blotassay. Since SFRPs have been shown to be transcriptionally inactivated incancer cells through promoter methylation, we determined the methylationstatus of the SFRP1and SFRP5promoters by methylation-specificPCR(MSP) analysis in the selected eight tumor samples. The resultsrevealed that SFRP1and SFRP5exhibited significantly higher levels of DNA methylation in HCC tumors compared with non--tumorous livertissues. Bisulfite-treated methylation-specific PCR (BSP) further confirmedthe results of this MSP analysis, showing the methylation of SFRP1andSFRP5is higher in the tumor tissues than in the surrounding nontumortissues. Deletion constructs of SFRP1and SFRP5promoters weresuccessfully constructed. In this research, we demonstrated that the strongestactivity area of SFRP1promoter is-407～-27nt and SFRP5promoter is-478~+47nt. HBx could inhibit SFRP1and SFRP5promoter activity.Over-expression of HBx could inhibit SFRP1and SFRP5promoter activity.And knock-down of Dnmt1or exogenous of SFRPs reduces hepatocellularproliferation in vitro through slowed cell cycle progression.Conclusion: Hepatitis B virus X protein reduce the promoter activity ofSFRP1and SFRP5through down-regulating the expression of SFRP1andSFRP5. HBx reduced the tumor cell proliferation and migration ability isrelated to activite DNA methyltransferase which activated theWnt/β-catenin signaling pathway.