Epigenetic repression of miR-132 expression by the hepatitis B virus x protein in hepatitis B virus-related hepatocellular carcinoma

Abstract

Hepatitis B virus x (HBx) protein is involved in the initiation and progression of HBV-related hepatocellular carcinoma (HCC) by regulating host protein-coding genes. However, the role of HBx in the epigenetic repression of miRNAs, which play important roles in gene regulation during hepatocarcinogenesis, remains largely unknown. In this study, the expression of miR-132 in HCC cells, HBV-related HCC tissues, and serum were determined using real-time PCR. The level of DNA methylation on the promoter of miR-132 was examined using methylation-specific PCR (MSP). MiR-132 was functionally characterized in HCC cells with transiently altered miR-132 expression. HBx-induced DNA hypermethylation of the promoter of miR-132 was found to be more prevalent in HBx-expressing HepG2 cells than in control cells. Consistently, MiR-132 expression was also more frequently down-regulated in HBV-related HCC tissues than in adjacent noncancerous hepatic tissues and had a significant inverse correlation with HBx expression in HBV-related HCCs. Serum miR-132 levels were found to be significantly correlated with levels in tumor tissue. Finally, proliferation and colony formation of HCC cells were found to be suppressed by miR-132-mediated inhibition of the Akt-signaling pathway in miR132 transfected cells. Our study has demonstrated the epigenetic repression of miR-132 expression through DNA methylation induced by HBx. This work provides novel mechanistic insights into HBV-mediated hepatocarcinogenesis and suggests that miR-132 may be a promising biochemical marker and may have therapeutic applications in HBV-related HCC.

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide [1]. Most cases of HCC are found in countries in which hepatitis B is endemic, such as those in Southeast Asia and sub-Saharan Africa. It has been reported that as much as 80% of HCC cases can be attributed to chronic HBV infection [2]. Previous studies have indicated that hepatitis B virus x (HBx), an oncoprotein encoded by HBV, can contribute to the development of HCC through multiple mechanisms [3], [4], [5]. Interestingly, recent studies have revealed that HBx may contribute to HBV-related HCC by interacting with de novo DNA methyltransferase 3A (DNMT3A) [6], suggesting that there may be a connection between DNA methylation and HBx in HBV-related HCC.

DNA methylation is almost exclusively found on 5C of cytosine of CpG dinucleotides. It takes place through enzymatic addition of methyl groups using DNMTs. It has long been thought as an inheritable, stable epigenetic mark related to long-term silencing and has been implicated in various biological processes, including gene imprinting, X chromosome and transposon repression, and the establishment and maintenance of stable cellular identities [7]. Aberrant DNA methylation has been associated with imprint-related diseases, neurological disorders, and cancer. For example, the promoters of tumor suppression genes have been shown to be frequently hypermethylated and silenced in various cancers [8]. The identification of epigenetic abnormalities, such as DNA hypermethylation on tumor suppressor genes, may provide biomarkers useful in cancer diagnostics. The correction of abnormal epigenetic modification with drugs also provides an opportunity a for cancer treatment. This approach may have a therapeutic advantage over genetic manipulation. In HBV-related HCC, various studies have shown which tumor-suppressor genes can be silenced by methylation of DNA promoters [9], [10], [11]. This suggests that DNA methylation may be suitable for use in the suppression of cancer-resistant genes in HCC.

MicroRNAs (miRNAs) are small, highly conserved non-coding RNAs. They suppress the expression of protein-coding genes by either translational repression or by mRNA degradation, depending on the manner in which they bind to complementary 3′-untranslated regions (3′-UTR) of target messenger RNA (mRNA) [12]. Recent reports have shown that miRNAs can function as oncogenes and as tumor suppressors to promote or prevent cancer development, respectively [13], [14]. Disease-related epigenetic transformations and cancer development have also been linked to aberrant expression of miRNAs [15]. In other cancers, miRNA levels decrease throughout the body [16]. This type of global miRNA repression has been observed in HCC, underscoring the protumorigenic effects of miRNA loss-of-function in cancer [17]. Previous studies have suggested that epigenetic modifications, particularly DNA hypermethylation, may play crucial roles in the initiation of HCC [18], [19]. Growing amounts of evidence show that miRNAs may act as targets and effectors of atypical DNA hypermethylation [20]. In human tumors, some miRNAs are inactivated by the aberrant hypermethylation of CGIs [21], [22]. However, DNA-methylation-associated silencing of tumor-suppressive miRNAs in HBV-related HCC remains poorly understood.

Previous studies have reported that some types of serum miRNAs may be useful as biochemical markers for human cancers [23], [24], [25]. We wondered if a similar situation occurs in HBV-related HCC and if some HBx-related miRNAs might be applied to diagnostics and therapeutics in HBV-related HCC.

In the present study, we show that miR-132 expression was down-regulated in HBx-expressing HCC cells relative to control HCC cells using real-time PCR. In particular, the mechanism by which miR-132 is silenced was found to be mediated by HBx-induced DNA methylation of the gene promoter. The miR-132 promoter was consistently found to be methylated. This was found to cause epigenetic repression of miR-132 expression and the miR-132 levels had a significant inverse correlation with HBx expression in HBV-related HCC tissues. In these tissues, serum miR-132 levels were found to be closely correlated with miR132 expression levels, suggesting that it may be useful as a diagnostic marker of HBV-related HCC. Further investigation showed that miR-132 could inhibit growth of hepatoma cells via the Akt-signaling pathway. Our results suggested that DNA-methylation-dependent inhibition of the tumor-suppressive function of miR-132 in HBV-related HCC may constitute a new avenue for cancer diagnostics and therapeutics.