Down-regulated microRNA-152 induces aberrant DNA methylation in hepatitis B virus–related hepatocellular carcinoma by targeting DNA methyltransferase 1

Authors

  • Jinfeng Huang,

    1. Department of Medical Genetics, Second Military Medical University, Shanghai, People's Republic of China
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  • Yue Wang,

    1. Department of Medical Genetics, Second Military Medical University, Shanghai, People's Republic of China
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  • Yingjun Guo,

    Corresponding author
    1. Department of Medical Genetics, Second Military Medical University, Shanghai, People's Republic of China
    • Department of Medical Genetics, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China
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    • fax: +86-21-81871053

  • Shuhan Sun

    Corresponding author
    1. Department of Medical Genetics, Second Military Medical University, Shanghai, People's Republic of China
    • Department of Medical Genetics, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China
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    • fax: +86-21-81871053


  • Potential conflict of interest: Nothing to report.

Abstract

The hepatitis B virus (HBV) X protein has been implicated as a potential trigger of the epigenetic modifications of some genes during hepatocarcinogenesis, but the underlying mechanisms remain unknown. MicroRNAs (miRNAs), which are noncoding RNAs that regulate gene expression, are involved in diverse biological functions and in carcinogenesis. In this study, we investigated whether some miRNAs are aberrantly expressed and involved in the regulation of the abnormal DNA methylation status in HBV-related hepatocellular carcinoma (HCC). Our results showed that the expression of microRNA-152 (miR-152) was frequently down-regulated in HBV-related HCC tissues in comparison with adjacent noncancerous hepatic tissues and was inversely correlated to DNA methyltransferase 1 (DNMT1) messenger RNA (mRNA) expression in HBV-related HCCs. The forced expression of miR-152 in liver cell lines resulted in a marked reduction of the expression of DNMT1 at both the mRNA and protein levels by directly targeting the 3′ untranslated regions of DNMT1. This in turn led to a decrease in global DNA methylation, whereas inhibition of miR-152 caused global DNA hypermethylation and increased the methylation levels of two tumor suppressor genes, glutathione S-transferase pi 1 (GSTP1) and E-cadherin 1 (CDH1). Conclusion: Our findings suggest that miR-152 is frequently down-regulated and regulates DNMT1 in HBV-related HCC. These findings support a tumor-suppressive role of miR-152 in the epigenetic aberration of HBV-related HCC and the potential development of miRNA-based targeted approaches for the treatment of HBV-related HCC. HEPATOLOGY 2010

Liver cancer is the fifth most common cancer in the world and the third most common cause of cancer-related death.1 Overall, 50% to 55% of cases of primary liver cancer are attributable to persistent hepatitis B virus (HBV) infections.2 HBV causes chronic infection in approximately 400 million people in the world.3 It is estimated that 50% of male carriers and 14% of female carriers will eventually die of the complications of cirrhosis and hepatocellular carcinoma (HCC).4 The hepatitis B virus X protein (HBx) has been implicated in HBV-related HCC pathogenesis and acts as a weak oncogene or a cofactor in hepatocarcinogenesis.5, 6 Several recent studies have suggested that HBx is also involved in epigenetic regulation during hepatocarcinogenesis.7, 8

Recent reports have emphasized that epigenetic modifications, especially DNA hypermethylation, might play crucial roles in the initiation of cancer. Methylation changes to the epigenome are controlled by DNA methyltransferases (DNMTs). Three catalytically active DNMTs have been identified in mammals: DNMT1, DNMT3A, and DNMT3B. Although the mechanisms leading to aberrant DNA hypermethylation remain to be fully elucidated, increased levels of DNMT1, DNMT3A, and DNMT3B have been observed in various malignancies, including leukemia, lung, colorectal, and breast tumors.9-12 It was reported that the average levels of messenger RNA (mRNA) for DNMT1 and DNMT3A were significantly higher in noncancerous liver tissues showing chronic hepatitis or cirrhosis versus histologically normal liver tissues. The levels were even higher in HCCs, and DNMT3B was significantly overexpressed in HCCs in comparison with the corresponding noncancerous liver tissues.13, 14 Increased protein expression of DNMT1 has been significantly correlated with the malignant potential and poor prognosis of human HCC.15 Moreover, the overexpression of HBx in vitro can increase total DNMT activity by the up-regulation of DNMT1 and DNMT3A.7 This suggests that DNMT overexpression contributes to gene promoter hypermethylation and in turn to HCC. However, the mechanism by which HBx activates DNMTs expression remains unknown.

MicroRNAs (miRNAs) are noncoding RNAs, 19 to 25 nucleotides long, that regulate gene expression by targeting mRNAs through base pairing at partially or fully complementary sites for cleavage or translational repression.16 Deviations from normal miRNA expression patterns play roles in human diseases, including cancers.17, 18 Some miRNAs may function as oncogenes or tumor suppressor genes (TSGs).19 Growing evidence supports a role for miRNAs as both targets and effectors in aberrant mechanisms of DNA hypermethylation. Some miRNAs have been reported to be inactivated in human tumors by the aberrant hypermethylation of CpG islands encompassing miRNA genes or located nearby.20, 21 It has also been reported that miRNAs are involved in the control of DNA methylation machinery. Fabbri et al.22, 23 recently demonstrated that miRNA-29b can target DNMT3s and induce aberrant DNA methylation in lung cancer and acute myeloid leukemia. We wondered if similar DNA methylation mechanisms occur in HCC and if there are some HBx-related miRNAs that can regulate DNMTs and then promote the aberrant DNA methylation.

We found that the expression of microRNA-152 (miR-152) was down-regulated in the livers of HBx transgenic mice in comparison with the livers of wild-type (WT) mice by miRNA microarray and real-time polymerase chain reaction (PCR) in our previous studies (see the supporting information in ref. 24). With in silico predictions, DNMT1 was defined as a potential target of miR-152. In this study, we tested whether miR-152 was down-regulated and regulated DNMT1 in HBV-related HCCs, and we measured the function of miR-152 in DNA methylation in vitro with the HCC cell lines. Our results showed that down-regulated miR-152 induced aberrant DNA hypermethylation by repressing expression of DNMT1 in HBV-related HCC.

Abbreviations:

CDH1, cadherin 1 type 1 E-cadherin; DNMT, DNA methyltransferase; EGFP, enhanced green fluorescent protein; GDM, global DNA methylation; GSTP1, http://www.genecards.org/cgi-bin/carddisp.pl?gene=GSTP1&search=GSTP1glutathione S-transferase pi 1; HBV, hepatitis B virus; HBx, hepatitis B virus X protein; HCC, hepatocellular carcinoma; LC-MS/MS, liquid chromatography–tandem mass spectrometry; miR-152, microRNA-152; miRNA, microRNA; mRNA, messenger RNA; Mut, mutated; PCR, polymerase chain reaction; RNAi, RNA interference; siRNA, small interfering RNA; TSG, tumor suppressor gene; UTR, untranslated region; WT, wild type.

Materials and Methods

Patient Samples.

The 20 HBV-related HCC tissues and the corresponding nearby noncancerous livers used in this study were obtained with informed consent from patients who underwent radical resection at Changhai Hospital (Second Military Medical University, Shanghai, China). The study was performed in accordance with the guidelines of the institutional review board of the Liver Cancer Institute.

Cell Culture and Transfection.

The liver cell lines HepG2, HepG2.2.15, Huh-7, LO2, and Hepa1-6 were obtained from the American Type Culture Collection. HepG2.2.15 and Huh-7 cells were cultured in minimum essential medium (Gibco-BRL) with 10% fetal bovine serum (Gibco BRL), and HepG2, LO2, and Hepa1-6 were cultured in Dulbecco's modified Eagle's medium (Gibco-BRL) with 10% fetal bovine serum (Gibco-BRL). Cells were maintained in a humidified 37°C incubator with an atmosphere of 5% CO2. Transfections were performed with a Lipofectamine 2000 kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Double-stranded miR-152 mimics, single-stranded miR-152 inhibitor, or their relative negative control RNA (GenePharma, Shanghai, China) at a final concentration of 50 nM was introduced into cells. Transfected cells were harvested at 24, 48, or 72 hours.

Inhibition of DNMT1 Expression.

The small interfering RNA (siRNA) sequences specifically targeting DNMT1 were synthesized by GenePharma as described.25 About 100 nM DNMT1 siRNA or control siRNA was transfected in HepG2 and Huh-7 cells by Lipofectamine 2000 as previously described by cell culture and transfection methods.

Construction of Vectors.

The 3′ untranslated regions (3′-UTRs) of DNMT1 containing an intact miR-152 recognition sequence were amplified by PCR from genomic DNA, and the PCR product was subcloned into a pGL3-promoter vector (Promega Corp., Madison, WI) immediately downstream of the luciferase gene. The primers used were 5′-GCTCTAGATCCCTGACACCTACCG-3′ (forward) and 5′-GCTCTAGACATAAAGTCTTAATTTCCACTC-3′ (reverse). A pGL3 construct containing the DNMT1 3′-UTR with point mutations in the seed sequence was synthesized with a QuikChange site-directed mutagenesis kit (Stratagene, Agilent Technologies, Palo Alto, CA). The hsa–miR-152 expression vector pcDNA3.1–hsa–miR-152 contains pri–miR-152 and some of its flanking sequences, and the sequences were cloned into a pcDNA3.1 vector (Promega). This vector can simulate the natural state of the stable expression of miRNA. The primers used were 5′-CCCTGACTCGAGGTGGACAC-3′ (forward) and 5′-GGGGCTGAAGTTCTGGGTC-3′ (reverse). The plasmid enhanced green fluorescent protein (pEGFP)–HBx vector was constructed in our laboratory previously.26 The complementary DNA encoding DNMT1 was PCR-amplified with PfuUltra II Fusion HS DNA polymerase (Stratagene) with the primers 5′-GGGGTACCATGCCGGCGCGTACCGC-3′ (forward) and 5′-GCGAATTCCTAGTCCTTAGCAGCTTCCTCCTCC-3′ (reverse) and was subcloned into the pcDNA3.1 vector. The resulting DNMT1 expression vector was confirmed by sequencing.

Reverse-Transcription Reaction and Quantitative Real-Time PCR.

Total RNAs were extracted with TRIzol reagent (Invitrogen). The first-strand complementary DNA was generated with a reverse-transcription system kit (Invitrogen). Stem-loop reverse transcription for mature miR-152 and U6 primers was performed as previously described.27 U6 RNA was used as an miRNA internal control. The primers used for stem-loop reverse-transcription PCR for miR-152 were as follows: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCAAGT-3′ (reverse transcription), 5′-GAGTGCTCAGTGCATGACAG-3′ (forward), and 5′-GTGCAGGGTCCGAGGT-3′ (reverse). Real-time PCR was performed with a standard SYBR-Green PCR kit protocol on a StepOne Plus system (Applied Biosystems, Foster City, CA). β-Actin was used as an endogenous control to normalize the amount of total mRNA in each sample. The primer sequences used were as follows: for mouse Dnmt1, 5′-CCCTTCCGAACCATCACC-3′ (forward) and 5′-CCAGCCGCACCTGTATGT-3′ (reverse); for human DNMT1, 5′-GCTACCTGGCTAAAGTCAAA-3′ (forward) and 5′-CCATTCCCACTCTACGG-3′ (reverse); for cadherin 1 type 1 E-cadherin (CDH1), 5′-CCGCCATCGCTTACA-3′ (forward) and 5′-GGCACCTGACCCTTGTA-3′ (reverse); and for glutathione S-transferase pi 1 (GSTP1), 5′-GCTGGAAGGAGGAGGTG-3′ (forward) and 5′-GGTGACGCAGGATGGTA-3′ (reverse). The real-time PCR reactions were performed in triplicate and included no-template controls. The relative expression was calculated with the comparative Ct method.

Luciferase Reporter Assay.

Cells (2 × 105) were cotransfected with 500 ng of pGL3-DNMT1-WT or pGL3-DNMT1-Mut constructs with miR-152 mimics or a negative control. Each sample was cotransfected with 50 ng of pRL-TK plasmid expressing renilla luciferase to monitor the transfection efficiency (Promega). A luciferase activity assay was performed 48 hours after transfection with the dual luciferase reporter assay system (Promega). The relative luciferase activity was normalized with renilla luciferase activity.

Western Blot Analysis.

Total cell lysate was prepared in a 1× sodium dodecyl sulfate buffer. Proteins in the same amount were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After incubation with antibodies specific for either DNMT1 (New England BioLabs, Beverly, MA) or β-actin (Cell Signaling Technology), the blots were incubated with goat anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized with enhanced chemiluminescence.

DNA Extraction and Bisulfite Treatment.

Genomic DNA was extracted from cells with the Axygen genomic DNA purification kit (Axygen Biotechnology, Hangzhou, China). Genomic DNA (0.5 μg) was treated with sodium bisulfite with the Zymo EZ DNA Methylation Gold kit (Zymo Research, Orange, CA) according to the manufacturer's instructions and then was subjected to further analysis.

Global DNA Methylation (GDM) Studies.

The GDM status of HepG2 and HepG2.2.15 cells after transfection with miRNA mimics or inhibitors was determined by the liquid chromatography–tandem mass spectrometry (LC-MS/MS) method as described previously.28

Bisulfite Genomic Sequencing.

DNA obtained at 72 hours from HepG2 cells transfected with the miR-152 inhibitor or miRNA inhibitor negative control were bisulfite-treated. Modified genomic DNA was then amplified with primers specific to the respective gene promoters by PCR. The primers used for detecting +227 and +601 of the GSTP1 promoter were 5′-GGATGGGGTTTAGAGTTTTTAGTATGG-3′ (forward) and 5′-CCTTCCCTACCAAACACATACTCCT-3′ (reverse); those for +117 and +356 of the CDH1 promoter were 5′-GGTTAGTTATGGGTTTTTGGAGTTGTAGT-3′ (forward) and 5′-CACCCCCCACTCCCATCACT-3′ (reverse). Bisulfite genomic sequencing PCR products were gel-extracted, subcloned into pMD-18T Vectors (Takara, Dalian, China), and transformed into Escherichia coli. Candidate plasmid clones were sequenced by Invitrogen, Ltd. (Shanghai, China).

Statistical Analysis.

The expressions of miR-152 in HCC patients were compared by the Wilcoxon signed-rank test. The relationship of miR-152 and DNMT1 mRNA expression was analyzed by Pearson's correlation. Bisulfite DNA sequencing results were compared by the Wilcoxon rank-sum test. The others were determined by the Student t test, and data are expressed as means and standard deviations from at least three independent experiments. All P values were two-sided and were obtained with the SPSS 13.0 software package (SPSS, Chicago, IL). A P value < 0.05 was considered statistically significant.

Results

miR-152 Is Significantly Down-Regulated in HBx Transgenic Mice and HCC Cell Lines Expressing HBx.

First, we assessed the aberrant expression of miR-152 in p21-HBx transgenic mice by both miRNA microarray and quantitative reverse-transcription PCR. We compared the miRNA profile of transgenic mouse liver tissues with WT mice of the same strain (C57BL/6), sex, and age (10 months old) and found that miR-152 was significantly down-regulated in transgenic mice (Fig. 1A).

Figure 1.

miR-152 was expressed at low levels in HBx transgenic mice and HCC cells expressing HBx. (A) The relative expression of miR-152 for 10-month-old p21-HBx transgenic mice versus WT mice was detected with microarrays and real-time PCR. (B) Relative expression of miR-152 in HepG2 and HepG2.2.15 cells by real-time PCR. (C) Relative expression of miR-152 in HepG2 cells with pEGFP or pEGFP-HBx (pHBx) transfection. Data are shown as means and standard deviations from triplicate experiments. *P < 0.05.

Next, we determined whether miR-152 was expressed differently in human HCC cells. The expression of miR-152 was markedly lower in the HepG2.2.15 cell line (a derivative of the human HepG2 hepatoma cell line that has been stably transformed with a head-to-tail dimer of HBV DNA) versus HepG2 cells (Fig. 1B). In order to verify whether this down-regulation correlated with the HBx expression, HepG2 cells were transiently transfected with pEGFP-HBx and pEGFP-N1. Forty-eight hours later, miR-152 was down-regulated in HBx-HepG2 cells in comparison with pEGFP-N1–transfected cells (Fig. 1C).

HBx Up-Regulates the Expression of DNMT1.

To investigate whether HBx alters DNMT1 expression, we measured the levels of DNMT1 mRNAs after the transient transfection of pEGFP-HBx into liver cancer cell lines, including HepG2 and Hepa1-6 (mouse hepatoma) cells. We found that DNMT1 was up-regulated in pEGFP-HBx–transfected cells in comparison with the pEGFP control groups (Fig. 2B). We also measured the DNMT1 mRNA level in HepG2 cells and HepG2.2.15 cells. The expression of DNMT1 was markedly higher in HepG2.2.15 cells versus HepG2 cells (Fig. 2A).

Figure 2.

DNMT1 was up-regulated in HepG2.2.15 cells and HCC cells expressing HBx. (A) Relative expression of DNMT1 mRNA in HepG2 and HepG2.2.15 cells by real-time PCR. (B) Relative expression of DNMT1 mRNA in HepG2 cells and Hepa1-6 cells with pEGFP or pEGFP-HBx (pHBx) transfection. Data are shown as means and standard deviations from triplicate experiments. *P < 0.05.

DNMT1 Is the Direct Target of miR-152.

As predicted by several in silico methods for target gene prediction, including PicTar,29 TargetScan,30 miRanda,31 and miRGen,32 the key enzyme in DNA methylation, DNMT1, was identified as one of the high-scoring candidate genes of miR-152 targets. As shown in Fig. 3A, the DNMT1-encoded mRNA contains a 3′-UTR element that is partially complementary to miR-152, and this indicates that miR-152 would directly target this site. To validate the miRNA-target interactions, the DNMT1 complementary sites, with or without mutations, were cloned into the 3′-UTR of the firefly luciferase gene and cotransfected with miR-152 mimics or negative control RNA in HepG2 cells. As shown in Fig. 3B, miR-152 significantly reduced the luciferase activity of the WT construct of the DNMT1 3′-UTR with respect to the negative control, whereas such a suppressive effect was not observed in cells with the Mut construct of DNMT1 3′-UTR. The miR-152 mimics at final concentrations of 50 and 100 nM reduced the luciferase activity, but there were no significant differences between the two groups. Therefore, miRNA at a final concentration of 50 nM was transfected into cells in the following experiments.

Figure 3.

miR-152 targets DNMT1. (A) WT and Mut 3′-UTRs of DNMT1, indicating the interaction sites between miR-152, and 3′-UTR of DNMT1. (B) Dual luciferase assay of HepG2 cells cotransfected with the firefly luciferase constructs containing the DNMT1 WT or Mut 3′-UTR and miR-152 mimics or scrambled oligonucleotides as the negative control. (C) DNMT1 mRNA expression after the transfection of pcDNA3.1–hsa–miR-152 or the control in HepG2.2.15 and LO2 cells. (D) DNMT1 mRNA expression after the transfection of the miR-152 inhibitor or miRNA inhibitor negative control in HepG2 and LO2 cells. (E) DNMT1 protein expression of HepG2.2.15 and HepG2 cells transfected with miR-152, miR-152 inhibitor, or their respective controls. Data are shown as means and standard deviations from at least three independent experiments. *P < 0.05.

miR-152 Represses DNMT1 Expression.

To test the hypothesis that miR-152 down-regulates DNMT1 in human liver cells, we transfected pcDNA3.1–hsa–miR-152 or pcDNA3.1 as the negative control into HepG2.2.15 cells and LO2 cells, and we transfected the miR-152 inhibitor or miRNA inhibitor negative control into HepG2 and LO2 cells. After 48 (RNA) or 72 hours (pcDNA3.1 vector) of transfection, we measured the mRNA and protein expression levels of DNMT1, respectively. Our results showed that enforced miR-152 expression led to a reduction of DNMT1 expression at both the mRNA and protein levels in comparison with the negative control in the two human liver cells (Fig. 3C,E). On the contrary, the inhibition of miR-152 increased the DNMT1 expression (Fig. 3D,E).

miR-152 Is Down-Regulated in Human HBV-Related HCC Tissues.

To determine whether miR-152 was expressed differentially in human primary liver cancer, we measured miR-152 expression levels in 20 pairs of human HBV-related HCC tissues and pair-matched normal liver tissues by real-time PCR. Among the 20 HBV-related HCC samples analyzed, the miR-152 levels were significantly decreased in 18 HCC samples (90%) in comparison with the adjacent noncancerous hepatic tissues (P = 0.00024, Wilcoxon signed-rank test; Fig. 4A).

Figure 4.

Expression of miR-152 and DNMT1 mRNA levels in 20 pairs of HBV-related HCC tissues and adjacent noncancerous hepatic tissues. (A) Down-regulated miR-152 expression in tumor tissues versus paired adjacent hepatic normal tissues from 20 HBV-related HCC patients by real-time PCR. The expression of miR-152 (log10 scale on the y axis) was normalized to U6 with respect to specimen NT1. Statistical differences were analyzed with the Wilcoxon signed-rank test (P = 0.00024). (B) The miR-152 and DNMT1 expression levels were inversely correlated in 20 HBV-related HCC samples. miR-152 and DNMT1 expression levels in these samples were measured by real-time PCR, and respective ΔCt values (miR-152 was normalized to U6; DNMT1 was normalized to β-actin) were subjected to a Pearson correlation analysis (r = −0.462, P = 0.02, Pearson's correlation).

miR-152 and DNMT1 mRNA Levels Are Reciprocally Regulated in Human HBV-Related HCC Tissues.

Next, we asked whether DNMT1 mRNA expression was inversely correlated with the levels of miR-152 in HBV-related HCC tissues. Twenty HCCs were analyzed for the expression levels of DNMT1 mRNAs and for miR-152 expression by real-time PCR. A statistically significant inverse correlation was observed between DNMT1 mRNA and miR-152 (n = 20, r = −0.462, P = 0.02, Pearson's correlation; Fig. 4B). These data showed the reciprocal regulation of the tumor suppressor miR-152 and its target DNMT1 in human HCCs and suggested that miR-152 may play a causal role in DNA methylation leading to liver cancer in chronic hepatitis B patients.

Changes in DNMT1 Expression Levels Do Not Affect miR-152 Expression in HCC Cells.

Because miR-152 was frequently down-regulated in HBV-positive HCC tissues in comparison with the adjacent noncancerous liver specimens, whereas DNMT1 expression appeared to be inversely correlated, we wondered if the reduction of miR-152 expression could be driven by the increase in DNMT1 expression. We enhanced or inhibited DNMT1 expression by transfecting the DNMT1 expression vector (pcDNA3.1-DNMT1) or DNMT1 siRNA into HepG2 and Huh-7 cells with the pcDNA3.1 vector and control siRNA, respectively, as the negative controls (Fig. 5A,B). After 48 hours of transfection, we measured the miR-152 expression levels of these cells and found that none of them were significantly changed in comparison with the negative controls (Fig. 5C,D).

Figure 5.

Changes in DNMT1 expression levels do not affect miR-152 expression in HCC cells. (A) DNMT1 mRNA and protein expression levels after the transfection of DNMT1 siRNA or control siRNA in HepG2 and Huh-7 cells. (B) DNMT1 mRNA expression levels after the transfection of the DNMT1 expression vector (pcDNA3.1-DNMT1) or the control in HepG2 and Huh-7 cells. (C) Expression levels of miR-152 after the transfection of DNMT1 siRNA or control siRNA in HepG2 and Huh-7 cells. (D) Expression levels of miR-152 after the transfection of pcDNA3.1-DNMT1 or pcDNA3.1 vectors in HepG2 and Huh-7 cells. Data are shown as means and standard deviations from at least three independent experiments. *P < 0.05.

Overexpression of miR-152 Reduces GDM.

We investigated whether the enforced expression of miR-152 could also functionally result in DNA hypermethylation. GDM was measured with an LC-MS/MS method in HepG2 and HepG2.2.15 cell lines after 72 hours of transfection with miR-152 inhibitor or miR-152 mimics, respectively. The miRNA negative control and inhibitor negative control served as negative controls for the mimics and inhibitor, respectively. We observed a reduction of 6.31% to 4.08% in GDM for the HepG2.2.15 cell line treated with miR-152 mimics in comparison with the negative controls and an increase of 4.55% to 5.88% in GDM for the HepG2 cell line treated with miR-152 inhibitors (Fig. 6A).

Figure 6.

The underexpression of miR-152 induces aberrant DNA hypermethylation in human liver cell lines. (A) GDM ratios of HepG2.2.15 and HepG2 cells after the transfection of miR-152 mimics, miR-152 inhibitor, or their respective controls. Genomic DNA was obtained from both cell lines after 72 hours of transfection, and GDM was measured by LC-MS/MS. (B) GSTP1 and CDH1 mRNA expression levels in HepG2 cells after the transfection of the miR-152 inhibitor or miRNA inhibitor negative control as measured by real-time PCR. (A,B) Data are shown as means and standard deviations from at least three independent experiments. *P < 0.05. Bisulfite DNA sequencing (bisulfite sequencing PCR) of (C) GSTP1 and (D) CDH1 promoters from HepG2 cells after 72 hours of transfection of the miR-152 inhibitor or miRNA inhibitor negative control (P < 0.005, Wilcoxon rank-sum test).

Underexpression of miR-152 Increases the DNA Methylation Levels of GSTP1 and CDH1 Promoter Regions in HepG2 Cells.

Several genes have been found to be silenced by DNA methylation induced by HBx via interactions with de novo DNMTs in HBV-HCC, including GSTP1 and CDH1. To assess whether inhibiting the expression of miR-152 could also lead to hypermethylation and silencing of these genes in HCC, we measured the DNA methylation levels of CDH1 and GSTP1 by bisulfate sequencing in the HepG2 cell line after transfection with the miR-152 inhibitor or miRNA inhibitor negative control. The GSTP1 DNA methylation level was increased in HepG2 cells transfected with the miR-152 inhibitor in comparison with the miRNA inhibitor negative control (mean of 85.8% versus 57.4%, P < 0.005, Wilcoxon rank test; Fig. 6C) Likewise, the CDH1 DNA methylation level was increased in HepG2 cells transfected with the miR-152 inhibitor in comparison with the control (mean of 23.8% versus 0%, P < 0.005, Wilcoxon rank-sum test; Fig. 6D). We also measured the mRNA levels of GSTP1 and CDH1 by real-time PCR in HepG2 cells after transfection. The GSTP1 mRNA expression level was significantly decreased in the miR-152 inhibitor group compared with the control group (Fig. 6B). This indicated that the inhibition of miR-152 could decrease GSTP1 expression by promoter DNA hypermethylation. However, the CDH1 mRNA level was not significantly changed after transfection, probably because the increase in the DNA methylation level was not sufficient to inhibit the mRNA expression.

Discussion

Epigenetic dysregulation of cellular genes is an integral feature in the development of human cancers. Increasing evidence has revealed that viral genes are some of the key players in regulating DNA methylation.33 The epigenetic mechanisms involved in virus-associated cancers are poorly understood, although aberrant promoter hypermethylation is a prevalent phenomenon in human cancers closely associated with viruses, such as HBV-related HCC. Hypermethylation is responsible for the silencing of TSGs involved in hepatocarcinogenesis. The involvement of the HBx protein in epigenetic regulation during hepatocarcinogenesis has been demonstrated previously, and it involves the activation of DNMTs7, 34 and the recruitment of DNMTs and methyl-CpG binding proteins to the target gene promoters. Interestingly, a strong correlation between HBV infection and epigenetic alterations of TSGs, including cyclin-dependent kinase inhibitor 2A (p16INK4a),35, 36 insulin-like growth factor binding protein 3,7 GSTP1,37 E-cadherin (CDH1),36, 38 and Ras association domain family 1A (RASSF1A),36 has been shown. However, how HBV affects the DNA methylation states remains unknown.

In this study, we characterized the role of miR-152 in the regulation of DNA methylation in HBV-related HCC for the first time. miR-152 induced aberrant DNA methylation by directly targeting DNMT1. Our data showed that miR-152 was frequently down-regulated in HBV-positive HCCs in comparison with corresponding noncancerous liver tissues. This indicated that miR-152 may have a tumor-suppressive role in HCC development. Our findings indicated that miR-152 expression was inversely correlated with DNMT1 expression in HCC; the down-regulation of miR-152, resulting in an up-regulation of DNMT1, was significant in HCC development. DNMT1 has been reported to be necessary and sufficient for maintaining global methylation and aberrant CpG island methylation in human cancer cells.39 Transcriptional silencing by CpG island methylation is a prevalent mechanism of TSG suppression in cancers. It is well known that decitabine, a potent and specific hypomethylating agent and an inhibitor of the DNMT activity that mediates DNA methylation, has been approved by the US Food and Drug Administration to treat myelodysplastic syndromes. Decitabine is also being studied in the treatment of cancer.40 miR-152 has the same effect in inhibiting DNMT; it may function as a tumor suppressor in HBV-related HCC development.

To our knowledge, this is also the first report showing that the inhibition of miR-152 results functionally in global DNA hypermethylation and increased methylation levels of the TSGs GSTP1 and CDH1 in HCC cell lines. The overexpression of miR-152 in HepG2.2.15 cells reduced GDM from 6.31% to 4.08%, whereas the miR-152 inhibitor in HepG2 cells increased GDM from 4.55% to 5.88%. The GSTP1 gene has been reported to be commonly epigenetically silenced by methylation in HBV-associated HCC, and somatic GSTP1 inactivation may contribute to the pathogenesis of this malignancy.35 In our study, the GSTP1 gene was demonstrated to be methylated in HepG2 cells, and the methylation level of its promoter that we detected was increased from 58.18% to 86.36% after transfection of the miR-152 inhibitor. CDH1 is also frequently silenced by methylation in HCC, and it has been reported that HBx can repress CDH1 expression by inducing the hypermethylation of its promoter.36, 38 In the current study, the methylation level of the CDH1 promoter region, which we measured, was increased from 0% to 23.8% in HepG2 cells. From these results, we can see that the TSG methylation levels increased, regardless of the initial methylation status. The relative mRNA level measurement showed that GSTP1 expression was significantly decreased after transfection of the miR-152 inhibitor in comparison with the control group, whereas the CDH1 mRNA level was not significantly changed. This probably occurred because the increasing DNA methylation level of the CDH1 promoter was not sufficient to inhibit the mRNA expression. The hypermethylation of CpG islands of TSGs promotes oncogenesis not only through transcriptional inactivation of these genes but also through the following mechanisms:

  • A signature C→T mutation in cancer cells: the cytosine residues in the methylated dinucleotide CpG have a higher mutation rate than the unmethylated cytosine.

  • The induction of chromosomal instability: aberrant DNA methylation leads to the genomic instability necessary for the development and progression of cancer, and DNA methylation is also correlated with allelic deletions.41, 42

Moreover, HBV DNA has been shown to contain CpG islands that can be methylated in human tissue both in a nonintegrated form43 and after integration into the human genome.44 The methylation of viral CpG islands can regulate viral protein production,45 which likely reflects viral adaptation to host cells. A DNA methylation–associated blockade of viral antigen presentation could help the virus to evade our immune system. The depletion of DNMT1 and DNMT3B by siRNA or upon treatment with the DNA demethylation agent caused DNA hypomethylation of the HBV genome in HCC cells.46 In the present study, we have demonstrated that HBx can up-regulate DNMT1 activity by inhibition of miR-152. These mechanisms may also be involved in the methylation of the HBV genome and the survival of HBV in host cells. Of course, this requires further experimental verification.

Furthermore, to gain insight into the biological function of miR-152 overexpression in HCC cells, the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, flow cytometric analysis, and the Transwell invasion assay were performed in the constructed cell lines (data shown in the supporting information). Ectopic miR-152 expression in HCC cells caused an inhibition of cell migration and invasion and induced cell apoptosis. However, we did not find any signification role for it in cell proliferation (Supporting Fig. 1). These results indicate that the enhanced expression of miR-152 by gene transfer could reverse the malignant phenotypes of HCC cell lines, and they suggest a tumor-suppressive role and a potential therapeutic target of miR-152.

We also believe that these finding have potentially relevant therapeutic implications. The results of this study provide a strong rationale for developing epigenetic therapies that use synthetic miR-152, alone or with other treatments, to reexpress the methylation-silenced TSGs and normalize aberrant patterns of methylation in HBV-related HCC.

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