Epigenetic Silencing Of Wnt Antagonists SFRPs By Hepatitis B Virus X Protein Plays An Important Role In Hepatocarcinogenesis

Objective: Hepatocellular carcinoma (HCC) is one of the mostcommon malignant tumor worldwide. Chronic hepatitis B virus (HBV)infection is one of the high risk factors associated with HCC in our country.Hepatitis B virus X protein (HBx), a pleiotropic protein encoded by HBVsmallest reading frame, plays a vital role in hepatocellular carcinogenesis,As a multifunctional regulator, HBx is capable of transactivation of manycellular oncogenes, or inactivation of tumor suppressor genes (such asc-myc, c-jun, p21, p53, etc) by interacting with series of nucleartranscription factors. Further, HBx is involved in the activation of severaloncogenic signal transduction pathways (such as Wnt, PI3K, JNK,MAPK/ERK, etc), which regulates cell proliferation, differentiation, cellcycle and other cellular biological behavior, thus eventually contribute tohepatocellular malignant transformation. However, the molecularmechanism that HBx involved in hepatocarcinogenesis is still unknown.Wnt signaling pathway is a higly evolved and conservative signalingin eukaryotes, which is implicated in a wide range of biological processes,such as in embryonic development and hemostasis of the adult stem cells. Abnormal activation of Wnt signaling pathway is closely related totumorigenesis, and it is reported that at least50%of HCC is associatedwith its aberrant activation. In addition to the genetic mutations such asCTNNB1(β-catenin), APC, AXIN1and AXIN2that directly activate theWnt signaling pathway, the dysregulation of other Wnt relevant regulatorscan also contribute to the abnormal activation of the pathway.Secereted frizzled related proteins (SFRPs), are the primaryextracellular signaling molecules that directly bind Frizzled receptor andantagonize the Wnt signaling pathway. Previous studies have suggested thatthe downregulation of SFRPs, which are resulted from hypermethylation ofits promoter region, may contribute significantly to hepatocarcinogenesis.Downregulation of SFRPs through promoter hypermethylation has beenidentified in a variety of human solid tumors, such as HCC, non-small celllung caner, gastric cancer, colon cancer. Recently, researchers havedemonstrated that viral coding protein, such as HCV core protein andNS5A protein, plays a critical role in epigenetic regulation of the host genestranscription, which is involved in cellular proliferation, differentiation andmigration, and enventually contributes greatly to the development of aseries of related disease. Whether HBx plays a direct role in epigeneticregulation of SFRPs remains unknown. Our previous study hasdemonstrated that the expression of SFRP1and SFRP5was downregulatedby HBx in hepatoma cell lines, which was dramatically restored by a 5-Aza-2’-deoxycytidine (DAC) treatment. In this study, we intend toanalyze the mechanism of HBx-induced SFRPs epigenetic silencing, and tofurther examine hepatoma cell proliferation and metastasis ability both invitro and in vivo by HBx-induced epigenetic silencing of SFRPs, thusexplore the concrete molecular mechanism in the pathogengesis ofHBV-related hepatocellular carcinoma.Methods:1) We used immuno precipitation (IP) and chromatinimmunoprecipitation (ChIP) experiments to observe the binding of DNAmethyltransferases (DNMTs) and histone deacetylases (HDACs) in SFRP1,5gene promoter regions induced by HBx, further we treated the hepatomacells with DAC and/or trichostatin A (TSA) and explored if the Aza and/orTSA treatment could reverse above epigenetic changes;2) Real-time PCRand statistical analysis were applied to explore the correlation of theexpression and methylation levels of SFRP1or SFRP5with DNMT1inHBV-HCC tissue samples;3) Besides, Western blot andimmunofluorescence assays were used to determine the effects ofknockdown of DNMT1or restoration of SFRP1and SFRP5on Wnt signalactivation and epithelial-mesenchymal transition (EMT) biomarkers inhepatoma cell lines which stably expresses HBx;4) Subsequently, Wedetermined wether RNA interference mediated-DNMT1inhibition orSFRP1and SFRP5restoration could attentuate the tumor growth and invasiveness in vivo, and further to explore its possible molecularmechanism.Results: Co-IP assay revealed that HBx can physically interact withDNMT1, DNMT3A and HDAC1. ChIP assay further confirmed that HBxinduced significantly enhanced recruitments of DNMT1, DNMT3A andmethyl-CpG binding protein (MBD) to SFRP1and SFRP5promoters,however, the expression of acetylated histone H3(AcH3) and the bindingof P300were decreased in above promoters. Besides, the recruitment ofDNMT1, MBD1and MBD2were greatly impaired in SFRP1and SFRP5promoters following DAC or TSA treatment. In contrast, the expression ofacetylated histone H3(AcH3) and the binding of P300were upregulated inabove promoters in hepatoma cells treated with DAC or TSA. DiminishedDNMT1, DNMT3A but enhanced histone H3acetylation was furtherstrengthened by the combination of Aza and TSA.In HBV-related hepatocellular carcinoma tissue samples, statisticalanalysis revealed that the expression levels of SFRP1and SFRP5weresignificantly downregulated in tumor tissues relative to paired adjacentnon-tumor tissues. Correlation analysis further demonstrated that SFRP1orSFRP5expression was inversely correlated with DNMT1expression, whiletheir methylation levels were positively correlated with DNMT1expression.Moreover, lower expressions of SFRP1and SFRP5were closely associatedwith poor tumor differentiation. By Western blot and immunofluorescence assays, we also found thatDNMT1silencing or SFRP1and SFRP5restoration could inhibit theexpression of Wnt signaling effector β-catenin and other relevant targetgenes, such as c-myc and cyclinD1. Moreover, the enhanced expression ofmesenchymal biomarkers Fibronectin and Vimentin, and the decreasedexpression of epithelial biomarker E-cadherin induced by HBx wererestored by knockdown of DNMT1or overecpression of SFRP1andSFRP5in hepatoma cells.In vivo, we demonstrated that silencing of endogenous DNMT1expression or exogenous expression of SFRP1and SFRP5significantlyattenuated the tumor growth. Moreover, the expression of Wnt effectorβ-catenin, cell growth biomarker proliferating cell nuclear (PCNA), andinvasion-related biomarkers VEGF and MMP9were effectively abrogatedby silencing of DNMT1or restoration of SFRP1and SFRP5.Conclusion: HBx facilitated the binding of DNMT1, DNMT3A andHDAC1to SFRP1and SFRP5promoters, and resulted in epigeneticrepression of SFRP1and SFRP5expression. HBx-induced repression ofSFRP1and SFRP5abnormally activate the Wnt signaling pathway andaccelerate the epithelial-mesenchymal transition (EMT) process, leading topromotion of cell proliferation and invasion in vitro and in invo, thuseventually contributes to HCC carcinogenesis. Our findings provide newinsights into HBV-associated hepatocarcinogenesis and may be helpful in developing specific inhibitors targeted to Wnt epigenetically silenced genesfor treating HCC.