Loss of E-cadherin is associated with acquisition of metastatic capacity. Numerous studies suggest histone deacetylation and/or hypermethylation of CpG islands in E-cadherin gene (CDH1) are major mechanisms responsible for E-cadherin silencing in different tumors and cancer cell lines. The Hepatitis B virus (HBV) encoded X antigen, HBx, contributes importantly to the development of hepatocellular carcinoma (HCC) using multiple mechanisms. Experiments were designed to test if in addition to CDH1 hypermethylation HBx promotes epigenetic modulation of E-cadherin transcriptional activity through histone deacetylation and miR-373. The relationships between HBx, E-cadherin, mSin3A, Snail-1 and miR-373 were evaluated in HBx expressing (HepG2X) and control (HepG2CAT) cells by western blotting, immunoprecipitation, chromatin immunoprecipitation as well as by immunohistochemical staining of liver and tumor tissue sections from HBV infected patients. In HepG2X cells, decreased levels of E-cadherin and elevated levels of mSin3A and Snail-1 were detected. Reciprocal immunoprecipitation with anti-HBx and anti-mSin3A demonstrated mutual binding. Further, HBx-mSin3A co-localization was detected by immunofluorescent staining. HBx down-regulated E-cadherin expression by the recruitment of the mSin3A/HDAC complex to the Snail-binding sites in human CDH1. Histone deacetylation inhibition by Trichostatin A treatment restored E-cadherin expression. Mir-373, a positive regulator of E-cadherin expression, was down-regulated by HBx in HepG2X cells and tissue sections from HBV infected patients. Thus, histone deacetylation of CDH1 and down-regulation of miR-373, together with the previously demonstrated hyper-methylation of CDH1 by HBx, may be important for the understanding of HBV-related carcinogenesis.
Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease (CLD) and hepatocellular carcinoma (HCC). HBV encoded X antigen, HBx, and pathways implicated in the self-renewal of stem cells contribute to HCC, but it is not clear whether HBx expression promotes “stemness.” Thus, experiments were designed to test the hypothesis that HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSCs). To test this hypothesis, HepG2 cells were stably transduced with HBx and then assayed for phenotypic and molecular characteristics of “stemness.” The relationship between HBx and “stemness”-associated markers was also evaluated by immunohistochemical staining of liver and tumor tissue sections from HBV infected patients. The results showed that Oct-4, Nanog, Klf4, β-catenin and EpCAM were activated by HBx in vitro and in vivo. EpCAM was detected in the nuclei of human HCC cells from infected patients. HBx promotes “stemness” by activating β-catenin and epigenetic up-regulation of miR-181, both of which target EpCAM. HBx expression was also associated with depressed levels of E-cadherin. Moreover, HBx stimulated cell migration, growth in soft agar, and spheroid formation. This work is the first to propose that HBV promotes “stemness” in the pathogenesis of HCC. HBx associated up-regulated expression of multiple “stemness” markers support the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristics of CSCs.
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