Epigenetic repression of miR-132 expression by the hepatitis B virus x protein in hepatitis B virus-related hepatocellular carcinoma
Highlights
► Demonstrate epigenetic-mediated miR-132 expression by HBx in HBV-related HCC ► Find serum miR-132 levels are significantly correlated with levels in tumor tissue ► Suggest miR-132 level may be suitable for use as biochemical marker of HBV-related HCC ► Implicate a potential application of miR-132 in HBV-related HCC targeted therapy
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.
Section snippets
Serum and tissue specimens
Serum and tissues (paired tissue specimens from HBV-related HCC tissues and adjacent noncancerous hepatic tissues) were obtained from patients undergoing surgical HCC resection. The specimens were collected at the Hepatobiliary Surgery Department of the First Affiliated Hospital of Chongqing Medical University, P.R. China. This study was approved by the Research Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.
Cell culture and transfection
The liver cell lines (HepG2, HepG2.2.15, and
Expression of miR-132 in HCC cells that express HBx
To determine whether miR-132 is regulated by HBx expression, we examined miR-132 expression levels in human HBx-expressing HCC cells and in control HCC cells in vitro using real-time PCR. MiR-132 expression was found to be significantly lower in HBV-DNA-transformed HepG2.2.15 cell line (transformed using a head-to-tail dimer) than in the control hepatoma-derived HepG2 cells (Fig. 1A). In order to determine whether HBx expression alone was sufficient to down-regulate miR-132 expression, the
Discussion
HBx is a multifunctional oncoprotein that plays many roles in the development of HCC. HBx alters the expression of the host protein-coding genes through its transactivating function, thus contributing to the initiation and progression of HCC. Epigenetic modulation of transcriptional activity of the target genes could be an important mechanism underlying HBx-mediated transformation. The fact that HBx does not bind to DNA is consistent with this. HBx has also been shown to elevate the overall
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
We thank Prof. Qian Tao (The Chinese University of Hong Kong, Hong Kong, China) for generously providing technical assistance. We also thank Prof. Tao Feng (Chongqing Medical University, P.R. China) for generously providing adenovirus vector. This study was supported by the National Natural Science Foundation of China (No. 81171562) and the Chongqing Health Bureau grant (No. 2011-1-010).
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Xufu Wei and Cui Tan have contributed equally to this paper.