Truncated HBx-dependent silencing of GAS2 promotes hepatocarcinogenesis through deregulation of cell cycle, senescence and p53-mediated apoptosis.

Hepatocellular carcinoma (HCC) is a worldwide threat to public health, especially in China, where chronic hepatitis B virus (HBV) infection is found in 80-90% of all HCCs. The HBV-encoded X antigen (HBx) is a trans-regulatory protein involved in virus-induced hepatocarcinogenesis. Although the carboxyl-terminus-truncated HBx, rather than the full-length counterpart, is frequently overexpressed in human HCCs, its functional mechanisms are not fully defined. We investigated the molecular function of a naturally occurring HBx variant which has 35 amino acids deleted at the C-terminus (HBxΔ35). Genome-wide scanning analysis and PCR validation identified growth arrest-specific 2 (GAS2) as a direct target of HBxΔ35 at transcriptional level in human immortalized liver cells. HBxΔ35 was found to bind the promoter region of GAS2 and attenuate its expression to promote hepatocellular proliferation and tumourigenicity. Further functional assays demonstrated that GAS2 induces p53-dependent apoptosis and senescence to counteract HBxΔ35-mediated tumourigenesis. Notably, GAS2 expression was significantly down-regulated in HCCs compared with the corresponding normal tissues. In conclusion, our integrated study uncovered a novel viral mechanism in hepatocarcinogenesis, wherein HBxΔ35 deregulates cell growth via direct silencing of GAS2 and thereby provides a survival advantage for pre-neoplastic hepatocytes to facilitate cancer development.

Carboxyl-terminal truncated HBx regulates a distinct microRNA transcription program in hepatocellular carcinoma development.

BACKGROUND: The biological pathways and functional properties by which misexpressed microRNAs (miRNAs) contribute to liver carcinogenesis have been intensively investigated. However, little is known about the upstream mechanisms that deregulate miRNA expressions in this process. In hepatocellular carcinoma (HCC), hepatitis B virus (HBV) X protein (HBx), a transcriptional trans-activator, is frequently expressed in truncated form without carboxyl-terminus but its role in miRNA expression and HCC development is unclear. METHODS: Human non-tumorigenic hepatocytes were infected with lentivirus-expressing full-length and carboxyl-terminal truncated HBx (Ct-HBx) for cell growth assay and miRNA profiling. Chromatin immunoprecipitation microarray was performed to identify the miRNA promoters directly associated with HBx. Direct transcriptional control was verified by luciferase reporter assay. The differential miRNA expressions were further validated in a cohort of HBV-associated HCC tissues using real-time PCR. RESULTS: Hepatocytes expressing Ct-HBx grew significantly faster than the full-length HBx counterparts. Ct-HBx decreased while full-length HBx increased the expression of a set of miRNAs with growth-suppressive functions. Interestingly, Ct-HBx bound to and inhibited the transcriptional activity of some of these miRNA promoters. Notably, some of the examined repressed-miRNAs (miR-26a, -29c, -146a and -190) were also significantly down-regulated in a subset of HCC tissues with carboxyl-terminal HBx truncation compared to their matching non-tumor tissues, highlighting the clinical relevance of our data. CONCLUSION: Our results suggest that Ct-HBx directly regulates miRNA transcription and in turn promotes hepatocellular proliferation, thus revealing a viral contribution of miRNA deregulation during hepatocarcinogenesis.