BackgroundThe 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.MethodsHuman 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.ResultsHepatocytes 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.ConclusionOur 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.

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.

1 Short-circuit current (SCC) technique was used to study the adrenoceptors involved in the electrogenic chloride secretion by cultured cauda epididymal epithelium of rats. Stimulation of the epithelium with noradrenaline (primarily PI-adrenoceptor selective agonist), salbutamol (132-adrenoceptor selective agonist) and adrenaline (non-selective P-adrenoceptor agonist) led to a rise in SCC. At a low chart-speed (2 mm min-'), the response profile to these agonists consisted of a peak followed by a sustained response considerably higher than the basal SCC. 2 The EC50s (doses of agonist producing 50% maximum response) of noradrenaline, salbutamol and adrenaline were 300, 115 and 10 nM respectively. Pretreating the tissues with 1 gM atenolol (PI-selective antagonist) and 10 AM butoxamine (132-selective antagonist) shifted the dose-response curves of noradrenaline (shifted EC50 = 4000 nM) and salbutamol (shifted EC50 = 1050 nM) to the right. Atenolol (1 LM) and butoxamine (10 LM) shifted the dose-response curve of adrenaline to the right with new EC50s of 30 nM and 115 nM, respectively. 3 The rapidly rising phase of the SCC response to noradrenaline and adrenaline observed at low chart-speed consisted of a brief and transient retraction followed by a rebound increase in SCC. At a high chart-speed (1 mm s-'), the retraction and rebound phenomenon manifested as a fast initial spike which could be blocked by phentolamine (non-specific x-adrenoceptor antagonist) in a dose-dependent fashion. Similar initial spikes were observed when the tissues were stimulated with phenylephrine (al-selective agonist) but not with isoprenaline (non-selective P-agonist) or forskolin (activator of adenylate cyclase). The response of the initial spike triggered by noradrenaline was dose-dependent and the EC50 was 2000 nM. 4 The present study showed that the electrogenic chloride secretion by rat epididymis could be stimulated by (xi-, 13I-and P2-adrenoceptor agonists. The al-mediated response had a faster onset and more transient action than the 3-counterpart. It is postulated that epididymal chloride secretion might be regulated by neural (noradrenaline-mediated) and humoral (adrenaline-mediated) controls and that the stimulus-secretion coupling mechanisms might involve both Ca2+ (al-mediated response) and adenosine 3':5'-cyclic monophosphate (P-mediated response) as intracellular second messengers.

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