Hepatitis B Virus X Protein (hbx) On The Regulation And Function Of The Notch1 And Snail

As early as 1970, Sherlock and his colleagues found chronic liver disease and primary liver-cell cancer with hepatitis-associated (Australia) antigen in serum, which indicated the association between chronic hepatitis B virus (HBV) infection and the development of hepatocellular carcinoma (HCC). In 1981, a powerful substantiation of the association between chronic HBV infection and HCC development was the result of a prospective cohort study in which Beasley and his colleagues followed 22, 707 Chinese men in Taiwan (China) and found that incidence of HCC among carriers of HBV surface antigen (HBsAg) is 63 times higher than among HBsAg non-carriers.Among all proteins encoded by HBV genome, hepatitis B virus X protein (HBx), a key nonstructural multifunctional regulatory protein of the virus, is at the intersection of HBV infection, replication, pathogenesis, and carcinogenesis. Although previous investigations have revealed that HBx might promote HBV-associated hepatocarcinogenesis through activating some oncogenic signal pathways, the role of HBx in the molecular mechanism for the development and progression of HBV-associated hepatocellular carcinoma (HBV-HCC) remains poorly understood.Although it has been reported that Notch1 signaling, as an evolutionary conserved local cell interaction mechanism, could exert a tumor suppressive function in hepatocarcinogenesis, the interaction between HBx expression and Notch1 signaling remains further elucidation. Besides, a high incidence of tumor recurrence and metastasis has been reported in hepatocellular carcinoma (HCC) patients with chronic hepatitis B virus (HBV) infection. Although the pathological relevance and significance of HBx in HBV-associated hepatocarcinogenesis attracted much attention in recent years, the role and molecular mechanism for HBx in tumor invasion and metastasis remain not fully understood.In the part one of this thesis, we report that HBx expression in hepatic and hepatoma cells resulted in decreased endogenous protein level of Notch1 intracellular domain (ICN1) and mRNA levels of its downstream target genes. These effects were shown due to reduction of Notch1 cleavage by HBx via suppressing presenilin1 (Psen1) transcription rather than inhibiting Notch1 transcription or its ligands expression. By transient HBx expression, decreased ICN1 resulted in enhanced cell proliferation, induced G1-S cell cycle progression, and blunted cellular senescence in vitro. Furthermore, the effect of blunted senescence-like growth arrest by stable HBx expression via suppressing ICN1 was shown in a nude mouse xenograft transplantation model. The correlation of inhibited Psenl-dependent Notchl signaling and blunted senescence-like growth arrest was also observed in HBV-associated HCC patient tumor samples. Our results revealed a novel function of HBx in blunting senescence-like growth arrest through decreasing Notchl signaling, which could be a putative molecular mechanism mediating HBV-associated hepatocarcinogenesis.In the part two of this thesis, we found that HBx expression could induce change of cell morphology from epithelial-like phenotype to mesenchymal-like phenotype, downregulation of epithelial marker expression (e.g. E-cadherin), nuclear translocation of P-catenin, upregulation of mesenchymal marker expression (e.g. N-cadherin, vimentin, and fibronectin), which indicates HBx expression could induce epithelial-mesenchymal transition (EMT) in hepatoma cells. This effect was shown due to increased EMT regulatory transcription factor Snail protein level by HBx transfection through enhancing Snail protein stability rather than upregulating Snail transcription, which could be reversed by siRNA Snail cotransfection with HBx. It was further elucidated HBx expression could enhance Snail protein stability via activating PI3K/AKT/GSK-3P signal pathway, which were substantiated by PI3K specific inhibitor LY294002 and Wortmannin treatment or constitutively active GSK-3βS9A mutant plasmid cotransfection with HBx. Moreover, it was also found that HBx expression in hepatoma cells could enhance cell migration ability and cell invasion ability in vitro, which could be reversed by siRNA Snail cotransfection, through wound healing assay, transwell cell migration assay, and collagen cell invasion assay. These results revealed a novel function of HBx in promoting EMT through stabilizing Snail protein via activating PI3K/AKT/GSK-3βsignaling, thus facilitating cell migration and invasion, which could provide a potential molecular mechanism for tumor invasion and metastasis in HBV-associated HCC patients.