Hepatitis B virus X protein promotes hepatocellular carcinoma transformation through interleukin-6 activation of microRNA-21 expression

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and chronic hepatitis B virus (HBV) infection is the major risk factor of HCC. The virus encodes HBV X (HBx) protein that plays a critical role in the development of HCC. Studies have revealed numerous HBx-altered genes and signalling pathways that heavily contribute to tumourigenesis of non-tumour hepatocytes. However, the role of HBx in regulating other critical gene regulators such as microRNAs is poorly understood, which impedes the exploration of a complete HBx-associated carcinogenic network. Besides, critical microRNAs that drive the transformation of non-tumour hepatocytes are yet to be identified. Here, we overexpressed C-terminal truncated HBx protein in a non-tumour hepatocyte cell line MIHA, and measured a panel of cancer-associated miRNAs. We observed that oncogenic miR-21 was upregulated upon ectopic expression of this viral protein variant. HBx-miR-21 pathway was prevalent in HCC cells as inhibition of HBx in Hep3B and PLC/PRF/5 cells significantly suppressed miR-21 expression. Subsequently, we showed that the upregulation of miR-21 was mediated by HBx-induced interleukin-6 pathway followed by activation of STAT3 transcriptional factor. The high dependency of miR-21 expression to HBx protein suggested a unique viral oncogenic pathway that could aberrantly affect a network of gene expression. Importantly, miR-21 was essential in the HBx-induced transformation of non-tumour hepatocytes. Inhibition of miR-21 effectively attenuated anchorage-independent colony formation and subcutaneous tumour growth of MIHA cells. Our study suggested that overexpression of miR-21 was critical to promote early carcinogenesis of hepatocytes upon HBV infection.