Recent findings have shown that miRNAs are often deregulated in HCC and significantly correlated with its clinicopathological features, such as cirrhosis, metastasis, recurrence, and prognosis 
. Functional studies further illustrate that miRNAs play important roles during hepatocarcinogenesis by directly influencing cell proliferation, apoptosis and metastasis of HCC cells 
. We and others have also delineated the miRNAs associated with HCC cells that were derived from chronic carriers of HBV and HCV, suggesting that miRNAs could be important mediators of HBV and HCV infection leading disease progression to HCC 
. However, the upstream mechanisms that contribute to the miRNA misexpression in HCC remain elusive. Here we provide evidence for miRNA deregulation in HCC through the HBV-encoded oncoprotein HBx. Using lentiviral-mediated expression system and integrative miRNA analysis, we found that full-length HBx and Ct-HBx distinctively regulated miRNA transcription in human hepatocytes. While full-length HBx activated and repressed similar numbers of miRNAs, its truncated form preferentially down-regulated miRNAs. Notably, at least 10 miRNAs were divergently regulated i.e. up-regulated by full-length HBx but down-regulated by the truncated counterpart. Our initial expression analysis in clinical samples implies that Ct-HBx at least partially contributes to the down-regulation of these miRNAs in human HCC, although further studies with larger sample size will be required to verify this notion.
Wang et al.
were first to systematically demonstrate the direct role of full-length HBx in deregulating miRNAs and explore the downstream mechanisms leading to hepatocarcinogenesis 
. In this study, seven and 11 miRNAs were found to be significantly up-regulated and down-regulated, respectively, in HBx-expressing HepG2 liver cancer cells compared to the control cells 
. Albeit using different culture and expression systems, our findings also indicated that the same subset of miRNAs (miR-125a, -193b and -99b) were up-regulated by full-length HBx in human hepatocytes and thus supported the authenticity of data. Furthermore, we found that a substantial number of full-length HBx-induced miRNAs have been reported to possess growth-inhibitory functions (). For example, miR-193b, frequently down-regulated in HCC 
, melanoma 
, breast 
and prostate cancers 
, was shown to promote apoptosis and inhibit growth of cancer cells 
. Concurred with the apoptosis-enhancing property of HBx 
, these findings suggested that miRNAs could be mediators of full-length HBx-triggered growth-suppression.
In sharp contrast, we demonstrated that Ct-HBx distinctively repressed a subset of growth-inhibitory miRNAs that were induced by its full-length counterpart ( and ). These findings provide mechanistic insight into how Ct-HBx abrogates full-length HBx-induced apoptosis 
and stimulates hepatocellular growth 
. Although HBx does not bind to DNA directly, it is capable to trans
-activate transcription elements in the nucleus 
. Recent studies have also shown that HBx achieves transcriptional suppression of important cancer-related genes e.g. TERT by enhancing promoter binding of transcription repressor like MAZ or physically interacting with the chromatin-modifying enzyme histone deacetylase 1 
. In the present study, we show for the first time that Ct-HBx binds to the promoters of growth-inhibitory miRNAs for transcriptional suppression (), presumably through its physical association with transcriptional repressors in the gene promoters 
. We speculate that this direct transcriptional regulation by Ct-HBx represents one of its major biological functions, since it has been shown to preferentially localize in the nucleus in contrast to the more cytoplasmic-orientated full-length counterpart 
. In addition, we demonstrated that full-length HBx bound to some miRNA promoters (Figure S2
). In some cases full-length HBx bound to similar promoter region with Ct-HBx and in other cases different or even no binding regions. The potential diverse cis
-regulatory modules (combination of transcription factor binding sites) in these distinct promoter regions and thus transcription factor partners 
might determine the regulatory function of HBx on miRNA transcription. In addition to miRNAs, our ChIP-chip analysis also demonstrated that Ct-HBx physically associates with promoters of protein-coding tumor-suppressors for gene silencing (Zhu, Cheng et al.
, unpublished data). Because these down-regulated genes may directly contribute to hepatocarcinogenesis, the molecular basis of such regulations e.g. the identity of specific transcriptional regulators and their functional interaction warrants further investigation.
As HBx is frequently integrated into the host genome in truncated form and over-expressed in HBV-associated HCC 
, Ct-HBx-mediated transcriptional repression may be one of the reasons underlying the reduced expressions of miR-26a and -29c in HCC cells ( and Figure S1
). One of the limitations of this study is the lack of enough HCC tissue samples for HBx protein expression analysis. In addition, the relatively small case numbers also hinders a solid conclusion to be drawn. Thus, we cannot exclude the possibility of the involvement of other mechanisms such as liver-enriched transcription factors in the transcriptional regulation of these miRNAs in human HCC 
. We demonstrated that the expression levels of miR-146a and -190, along with miR-26a and -29c, were significantly lower in HCCs compared to the matching non-tumor tissues (). Together with the reported down-regulation of miR-30d and -193b in other malignancies 
, it is conceivable that specific miRNA repression is vital for Ct-HBx-mediated hepatocarcinogenesis. For example, viral-mediated administration of miR-26a in a mouse model of HCC resulted in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression 
. We and others have recently demonstrated that miR-26a possessed the tumor-suppressive functions by directly targeting the EZH2 oncogene in cancers e.g. nasopharyngeal carcinoma 
, lymphoma 
and HBV-associated HCC cells (Figure S3
). Moreover, we found that the histone methyltransferase EZH2 
was over-expressed in human HCCs and promoted HCC cell growth and tumorigenicity at least partially through activation of Wnt/β-catenin signaling 
. Therefore, Ct-HBx might promote HCC development via the deregulation of miR-26a control on the EZH2 epigenetic machinery 
. This notion is further supported by findings from large cohorts of HBV-associated HCCs demonstrating that patients whose tumors had low miR-26a expression had shorter overall survival than those with high tumor miR-26a expression 
Another important miRNA transcriptionally repressed by Ct-HBx was miR-29c, whose down-regulation was significantly associated with worse disease-free survival of HCC patients 
. This miRNA has been shown to repress HCC growth in vitro
and in vivo
through promotion of apoptosis by targeting anti-apoptotic molecules, Bcl-2 and Mcl-1 
. Apart from apoptosis pathway, we and others have also determined the roles of miR-29 in controlling differentiation, DNA methylation and p53 pathways via repression of YY1 
, DMNT3A/3B 
and CDC42 and p85 alpha 
, respectively. Furthermore, miR-146a, -193b and -29c have been shown to inhibit tumor cell invasiveness and metastatic potential by repressing EGFR, urokinase-type plasminogen activator and extracellular matrix proteins, respectively 
. Taken together, Ct-HBx, via its repressed miRNAs and the corresponding perturbed gene-networks, may not only cause uncontrolled growth placing large numbers of cells susceptible to neoplastic transformation but also promote tumor progression.
In conclusion, this study uncovers the role of HBx, especially the naturally occurring carboxyl-terminal truncated mutant, in regulating cellular miRNAs of human hepatocytes. In contrast to its full-length counterpart, Ct-HBx distinctively down-regulated a set of growth-inhibitory miRNAs and concordantly promoted the growth of hepatocytes. Our integrative miRNA profiling and ChIP-chip analysis also highlights the nuclear trans-repressor role of Ct-HBx in miRNA regulation. Together with the miRNA expression analysis in clinical specimens, our findings suggest that Ct-HBx, at least in part, drives the miRNA transcriptional program in HCC development and represents a new therapeutic target for HCC treatment.