EZH2-mediated epigenetic silencing of tumor-suppressive let-7c/miR-99a cluster by hepatitis B virus X antigen enhances hepatocellular carcinoma progression and metastasis

Background: Hepatitis B virus (HBV)-encoded X antigen, HBx, assists in the development of hepatocellular carcinoma (HCC) through complex mechanisms. Our results provide new insights into the EZH2 epigenetic repression of let-7c that promotes HCC migration induced by HBx. Thus, let-7c and HMGA2 represent key diagnostic markers and potential therapeutic targets for the treatment of HBV-related HCC. Results: We investigated the epigenetic regulation of let-7c, an important representative miRNA in liver tumor metastasis, in human HCC cells to verify the effect of HBx. Based on quantitative PCR (qPCR) of mRNA isolated from tumor and adjacent non-tumor liver tissues of 24 patients with HBV-related HCC, EZH2 expression was significantly overexpressed in most HCC tissues (87.5%). We executed a miRNA microarray analysis in paired HBV-related HCC tumor and adjacent non-tumorous liver tissue from six of these patients and identified let-7c, miR-199a-3p, and miR-99a as being downregulated in the tumor tissue. Real-time PCR analysis verified significant downregulation of let-7c and miR-99a in both HepG2X and Hep3BX cells, which stably overexpress HBx, relative to parental cells. HBX enhanced EZH2 expression and attenuated let-7c expression to induce HMGA2 expression in the HCC cells. Knockdown of HMGA2 significantly downregulated the metastatic potential of HCC cells induced by HBx. Conclusions: The deregulation of let-7c expression by HBx may indicate a potential novel pathway through deregulating cell metastasis and imply that HMGA2 might be used as a new prognostic marker and/or as an effective therapeutic target for HCC.