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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

MicroRNAs (miRNAs) are highly conserved small noncoding RNAs participating in regulation of various cellular processes. Viruses have been shown to utilize cellular miRNAs to increase their replication in host cells. Until now, the role of miRNAs in hepatitis B virus (HBV) replication has remained largely unknown. In this study, a number of miRNA mimics were transfected into hepatoma cell lines with HBV replication. It was noted that microRNA-1 (miR-1) transfection resulted in a marked increase of HBV replication, accompanied with up-regulated HBV transcription, antigen expression, and progeny secretion. However, bioinformatics and luciferase reporter analysis suggested that miR-1 may not target the HBV genome directly but regulate the expression of host genes to enhance HBV replication. Further studies showed that miR-1 was able to enhance the HBV core promoter transcription activity by augmenting farnesoid X receptor α expression. In addition, miR-1 arrested the cell cycle at the G1 phase and inhibited cell proliferation by targeting histone deacetylase 4 and E2F transcription factor 5. Analysis of the cellular gene expression profile indicated that miR-1 transfected hepatoma cells developed a differentiated phenotype of hepatocytes. Conclusion: MiR-1 regulates the expression of several host genes to enhance HBV replication and reverse cancer cell phenotype, which is apparently beneficial for HBV replication. Our findings provide a novel perspective on the role of miRNAs in host-virus interactions in HBV infection. (HEPATOLOGY 2011;)

MicroRNAs (miRNAs) are endogenous, noncoding RNAs of lengths of 20 to 25 nucleotides that are emerging as key players in regulating gene expression in eukaryotes, influencing various biological processes like development, infection, immunity, and carcinogenesis.1, 2 MiRNAs are engaged in either translational arrest or degradation of targeted transcripts through imperfect base pairing with the 3′-untranslated region (UTR) of the target transcripts. Intriguingly, miRNAs may also lead to an up-regulation of gene expression by targeting 5′UTR3, 4 or under cell cycle arrest conditions.5 Functional messenger RNA (mRNA) targets are generally fully complementary to nucleotides 2-8 at the 5′ end of the miRNA, referred to as the miRNA “seed region.”6 Bioinformatic analyses have suggested that a single miRNA may suppress a number of genes and each mRNA can be targeted by multiple miRNAs.7 It is estimated that more than 30% of human genes are regulated by miRNAs.8

At present, the roles of cellular miRNAs in viral life cycles are under investigation. Recent findings highlighted that miRNAs of host cells may affect viral gene expression in direct or indirect manners and may play a significant role in maintenance of viral replication, latency, and evasion of the host immune system.9 For example, the human miR-32 inhibits the accumulation of primate foamy virus type 1 in host cells.10 In contrast, miR-122 facilitates hepatitis C virus (HCV) replication by binding to the 5′ end of the viral genome.3 Additionally, interferon beta (IFN-β) was reported to modulate the expression of several cellular miRNAs to inhibit HCV replication.11

Hepatitis B virus (HBV) is an enveloped DNA virus of the family Hepadnaviridae and causes acute and chronic hepatitis in humans.12 HBV replication is dependent on host cell proliferation status13 and controlled by a variety of cellular transcription factors, in particular, several nuclear receptors like farnesoid X receptor α (FXRA), hepatocyte nuclear factor 4α (HNF4A), liver X receptor (LXR), retinoid X receptor α (RXRA), and peroxisome proliferator activated receptor α/γ (PPARA/G).14, 15 Recent studies have shown that miRNA expression profiles could be affected by HBV infection in HBV-related hepatocellular carcinoma.16 Some miRNA-regulated genes related to immune response, cell death, DNA damage and recombination, and transcription showed a decreased expression pattern, suggesting the involvement of miRNAs in HBV infection and HBV-related carcinogenesis.16

Considering the possibility that cellular miRNAs may play an important role in HBV pathogenesis, a number of miRNAs were selected and their effect on HBV replication was examined in HBV-expressing hepatoma cell lines. Our data suggest that miR-1 is able to regulate the expression of multiple cellular genes, inhibit cell proliferation, and promote cell differentiation, resulting in the enhancement of HBV replication in hepatoma cells. Our results provide a new concept to understand the role of miRNAs in HBV replication and present a useful way to identify host factors involved in HBV life cycle.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Reagents.

All miRNAs and siRNAs used in the present study are listed in Supporting Information Table 1. An HBV replication-competent clone pSM2 harboring a head-to-tail tandem dimer of the HBV genome (GenBank accession number: V01460) was provided by Dr. Hans Will (Heinrich-Pette-Institute, Hamburg, Germany). The expression plasmid encoding full length human HDAC417 was purchased from Addgene (Cambridge, MA). The class I histone deacetylases inhibitor trichostatin A (TSA), FXRA antagonist guggulsterone (GGS), cell cycle synchronization chemicals aphidicolin and nocodazole were purchased from Sigma-Aldrich (Steinheim, Germany).

Cell Culture and Transfection.

Human hepatoma cell lines HepG2 and Huh7 were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin and maintained at 37°C in a humidified 5% CO2 atmosphere. HepG2.2.15 cells with integrated dimers of the HBV genome (GenBank accession number: U95551) and Con-1 cells with a subgenomic HCV replicon (kindly provided by Prof. Dr. Ralf Bartenschlager, University of Heidelberg, Germany) were cultured with 500 μg/mL of G418 (Sigma-Aldrich). Primary human hepatocytes were isolated from liver transplantation donor by perfusion and cultured as described.18 Plasmids, miRNAs, and small interfering RNAs (siRNAs) were transfected into cells at indicated concentrations using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Analysis of HBV Replication and Gene Expression.

HBV replicative intermediates (HBV RI) from intracellular core particles and HBV transcripts were extracted from hepatoma cell lines and detected by southern and northern blot, respectively, according to the published protocols.19 HBV progeny DNA was extracted from cell culture supernatants using QiAamp DNA Blood Mini kit (Qiagen) and quantified by real-time polymerase chain reaction (PCR) as described.20 HBV RNAs in cells were also detected using quantitative real-time reverse transcriptase (RT)-PCR assay (primer sequences are listed in Supporting Information Table 2). A monoclonal antibody (clone 10E11, Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect hepatitis B c-antigen (HBcAg) expression by western blot as described below. The levels of HBsAg and HBeAg in culture supernatants were determined using the Architect system and HBsAg and HBeAg CMIA kits (Abbott Laboratories, Wiesbaden-Delkenheim, Germany) according to the manufacturer's instructions.

Western Blot Analysis.

Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted with primary antibodies recognizing histone deacetylase 4 (HDAC4), phosphorylated retinoblastoma (p-Rb), and albumin (ALB) (Cell Signaling Technology); FXRA (R&D Systems, Minneapolis, MN); E2F transcription factor 5 (E2F5) (Santa Cruz Biotechnology), and β-actin (Sigma-Aldrich), respectively. Protein bands were visualized using ECL Plus Western blotting detection reagents (Amersham Biosciences, Buckinghamshire, UK) as described.18

Real-Time RT-PCR Assay.

Total RNA was extracted with TRIzol (Invitrogen) including a digestion with DNase Set (Qiagen). The expression of different cellular genes was determined by quantification of specific mRNAs using commercial Quantitect Primer Assays (Qiagen, primer sequences not available). The real-time RT-PCR was performed by a one-step method with 100 ng of total RNA using QuantiFast SYBR Green RT-PCR Kit (Qiagen) on a Light Cycler (Roche Diagnostics), as described.18 For each sample, RT-PCR was performed in duplicate. The expression levels of each gene are presented as values normalized against 106 copies of β-actin transcripts.

Vector Construction and Luciferase Reporter Assay.

The luciferase reporter vectors pSP1, pSP2, pCP, pXP, pEN2/CP, pEN2/CP-EmCm, and pmiR-E2F5-3UTR were generated and luciferase reporter assays were performed as described in the Supporting Information Materials and Methods.

Cell Proliferation and Cell Cycle Analysis.

Cell proliferation was determined using the Cell Proliferation reagent kit I (WST-1; Roche Diagnostics) and 3H-thymidine incorporation assay as described.21 For cell cycle analysis, HepG2.2.15 cells were transfected with 20 nM of miR-1 or control miRNA (miR-C), cultured for 48 hours, then treated with or without 4 μg/mL of aphidicolin or 100 nM of nocodazole for an additional 24 hours and fixed in the presence of 70% ethanol at 4°C. After washing, fixed cells were incubated in phosphate-buffered saline (PBS) containing 20 μg/mL of propidium iodide, 200 μg/mL of RNase A, and 0.1% Triton X-100 (BD Biosciences, Bedford, MA) at 37°C for 20 minutes. The stained cells were then analyzed for cell cycle distribution with a flow cytometer (FACScaliber, Becton Dickinson).

Microarray Analysis.

Total RNA was isolated from HepG2.2.15 cells transfected with miR-1 and control miRNA and subjected to microarray analysis using the Affymetrix Human Genome U133A Plus 2.0 Array according to the manufacturer' instructions. Differentially expressed genes were identified using Student's t test on log-transformed data and represented as heatmap by Spotfire (TIBCO Software, Somerville, MA). These genes were further subjected to Gene Set Enrichment Analysis (GSEA) to identify the biological patterns of the genes. The significance threshold for the permutation test was set at P < 0.05.

Statistical Analysis.

The statistical analysis was carried out using GraphPad (San Diego, CA). Analysis of variance with Student's t test was used to determine significant differences in multiple comparisons. P < 0.05 was considered statistically significant. Representative data from a series of at least three experiments are shown. Data are presented as standard error of the mean (SEM).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Identification of miR-1 as a Candidate for Regulation of HBV Replication.

To identify miRNA candidates influencing HBV replication, several miRNA mimics (Sequences information are in Supporting Information Table 1) which are functionally related to cell differentiation, viral infection, innate immune response, hypoxia stress, and cancer were selected and transfected into HepG2.2.15 cells at a concentration of 20 nM. HBV RI were isolated at day 4 after transfection and analyzed by southern blot. As compared to control miR-C, HBV replication was significantly enhanced and quantitative real-time PCR showed 3.5-fold up-regulation of HBV RI by miR-1 transfection (Fig. 1A, lane 3). MiR-146 and miR-214 increased HBV replication slightly, whereas miR-210 decreased HBV replication by about 40%. The liver specific miRNA-122, as well as miR-132, did not have a significant effect on HBV replication (Fig. 1A). In Con 1 cells with HCV subgenomic replicon, miR-122 transfection resulted in an increase of HCV RNA copy number,3 whereas other miRNAs had no significant influence on HCV replication (Supporting Information Fig. 1). To further evaluate these miRNAs effects on HBV replication, miRNAs and a replication competent clone of HBV pSM2 were cotransfected into Huh7 and HepG2 cells. Consistently, the amount of HBV RI was increased about 2.5-fold after miR-1 transfection (Fig. 1B). Other miRNAs tested so for had no significant effect on HBV replication in the cotransfection system (data not shown). Based on these results, we subsequently focused on miR-1.

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Figure 1. Identification of miR-1 as a candidate for regulation of HBV replication. (A) HepG2.2.15 cells were transfected with different miRNA mimics and a nonspecific control miR-C at 20 nM and cultured for 4 days. Mock transfection was included as a control. Cells were harvested at day 4 and the levels of HBV RI were determined by southern blot (upper panel) and real-time PCR analysis (bottom panel). The positions of relaxed circular (RC), double-stranded linear (DL), and single-stranded (SS) DNAs were indicated in the southern blot. The values obtained from mock-transfected samples were set at 1.0. (B) Hepatoma cell lines Huh7 (left) and HepG2 (right) were cotransfected with 1.5 μg of pSM2 combined with 20 nM of miR-1 or miR-C. Cells were harvested at day 3 after transfection and the levels of HBV RI were determined as described above. The values obtained from miR-C transfection were set at 1.0. *P < 0.05; **P < 0.01; NS, statistically not significant.

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Up-Regulation of HBV Transcription, Gene Expression, and Progeny Secretion by miR-1.

Next we examined the effect of miR-1 on the different stages of the HBV life cycle. HepG2.2.15 cells were transfected with miR-1 at concentrations ranging from 0.1 to 40 nM. An increased HBV replication was observed at a low concentration of 0.1 nM of miR-1 and up to 5.0-fold at 40 nM (Fig. 2A; Supporting Information Fig. 2A). The up-regulation of HBV replication by miR-1 transfection became recognizable after 2 days, increased with time, and maintained at least up to 14 days (Supporting Information Fig. 2B). HBV RNA levels were elevated significantly as determined by real-time RT-PCR (Fig. 2B, upper panel) and northern blot (Fig. 2B, bottom panel). The amount of HBV progenies released into culture supernatants was also increased in a dose-dependent manner (Fig. 2C). The amount of HBsAg in culture supernatants of transfected HepG2.2.15 cells increased in a similar manner after miR-1 transfection. A slight increase of HBeAg could be observed after transfection with 40 nM of miR-1, whereas HBcAg expression in cytoplasm was clearly increased, as shown by western blot (Fig. 2D). Transfection with miR-C had no influence on the levels of HBV RI, RNAs, proteins, and the production of HBV progenies (Fig. 2A-D). These results confirmed that miR-1 effectively enhanced HBV replication, as well as transcription, gene expression, and progeny viral secretion.

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Figure 2. Up-regulation of HBV transcription, gene expression, and progeny secretion by miR-1. HepG2.2.15 cells were transfected with miR-1 at different concentrations (0, 5, 10, 20, and 40 nM) and with 40 nM of miR-C. Cell lysates and culture supernatants were harvested at day 4 after transfection. (A) HBV RI was determined by southern blot (upper panel) and real-time PCR (bottom panel) analyses. The values obtained from mock-transfected samples were set at 1.0. (B) The levels of HBV RNAs were determined in three parallel samples relative to β-actin by real-time RT-PCR (upper panel) and the representative samples were subjected to northern blot analysis by the agarose-formaldehyde method. 18S and 28S RNA were used as loading control (bottom panel). (C) HBV progeny DNA in supernatant was quantified by real-time PCR. (D) The levels of secreted HBsAg and HBeAg in culture media were measured by CMIA test (upper panel). The intracellular HBcAg expression was analyzed by western blot assay with β-actin as the loading control (bottom panel). *P < 0.05.

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Sequence-Dependent Effect of miR-1 Action on HBV Replication.

To investigate molecular mechanisms of miR-1 effect on HBV replication, we first addressed the sequence specificity of the effect of miR-1. A mutated miR-1 (m-miR-1) with a mutated seed sequence (Fig. 3A) and miR-206, an miRNA with the identical miR-1 seed-sequence but a different sequence at its 3′ end, were used for comparison with miR-1. Transfection of HepG2.2.15 cells with m-miR-1 and miR-206 did not enhance HBV replication (Fig. 3A). Further, cotransfection of miR-1 and its specific antisense inhibitor anti-miR-1 abolished the increase of HBV RI in HepG2.2.15, whereas the enhancing effect of miR-1 on HBV RI remained unchanged if an unrelated anti-miR-C was cotransfected (Fig. 3B, lane 3). Consistently, knockdown of argonaute-2 (Ago2), a main component of RNA-induced silencing complex, by specific siRNA appeared to attenuate the effect of miR-1 (Fig. 3C, lane 4). These results suggested that up-regulation of HBV replication was mediated by miR-1-guided RISC formation.

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Figure 3. The sequence-specific effect of miR-1 action on HBV replication. HepG2.2.15 cells were transfected with 20 nM of m-miR-1 and miR-206 (A), or cotransfected with 50 nM of anti-miR-1 and anti-miR-C (B), or cotransfected with 20 nM of Ago-2 siRNA and control siRNA (C). Cells were harvested at day 4 after transfection and the levels of HBV RI were determined by southern blot (upper panel) and real-time PCR (bottom panel) analysis. A comparison of the sequences of miR-1, miR-206, and m-miR-1 is given in part A. *P < 0.05; NS, statistically not significant.

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A critical feature of a direct interaction between miRNAs and target mRNAs is the presence of the corresponding seed sequences in the target.2 However, the complementary sequence (ACATTCC) of miR-1 seed sequence which was required for its binding to target mRNA was not found in the HBV genomic sequence. Consistently, cotransfection of pMIR-REPORT system with cloned full length or four fragments of HBV genome and miR-1 into HepG2 cells did not result in a decrease of luciferase gene expression (Supporting Information Fig. 3). Taken together, the data suggest that it is unlikely that miR-1 regulates HBV gene expression and replication by a direct interaction with genomic sequence of HBV.

HDAC4 Is Involved in miR-1 Action on HBV Replication.

These results suggested that miR-1 may act on specific cellular targets and thereby enhances HBV replication and gene expression in an indirect manner. Previously, a member of class II histone deacetylase (HDAC4) was identified as a cellular target of miR-1.22 Similarly, transfection with miR-1 led to a markedly reduced expression level of HDAC4 protein in HepG2.2.15 cells (Fig. 4A). The reduction of HDAC4 by miR-1 hinted at the potential role HDAC4 on HBV replication, similar to the recent results of HDAC1.23 Indeed, the knockdown of HDAC4 expression by specific siRNAs led to nearly a 2.5-fold increase in HBV replication in HepG2.2.15 cells (Fig. 4B), as well as the use of broad-spectrum HDAC inhibitor TSA (Supporting Information Fig. 4). Furthermore, cotransfection of an HDAC4 expression vector pHDAC4 with miR-1 could attenuate the increased replication of HBV (Fig. 4C). We concluded that HDAC4 is a target of miR-1 and may play a significant role in the action of miR-1 on HBV replication.

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Figure 4. HDAC4 is involved in miR-1 action on HBV replication. HepG2.2.15 cells were transfected with 10 and 20 nM of miR-1 or miR-C for 4 days. HDAC4 protein levels were assessed by western blot with β-actin as the loading control. (B) HepG2.2.15 cells were transfected with siRNA specific for HDAC4 and siRNA-C at 20 nM and cultured for 4 days. HBV RI was determined by southern blot (middle panel) and real-time PCR (bottom panel) analyses. HDAC4 protein levels were assessed by western blot (upper panel). (C) HepG2.2.15 cells were cotransfected with 20 nM of miR-1 and 1 μg/mL of HDAC4 expression vector (pHDAC4), or control plasmid (pcDNA3.1) for 4 days; the levels of HBV RI were determined as described above. *P < 0.05.

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MiR-1 Transactivates the HBV Core Promoter by Augmenting FXRA Expression.

The modulation of HDAC4 expression by miR-1 may lead to changes of HBV promoter activity. Thus, four pGL3-based luciferase reporter constructs pSP1, pSP2, pCP, and pXP containing the region of HBV SP1, SP2, core, and X promoters were cotransfected with miR-1 into HepG2.2.15 cells. The ectopic expression of miR-1 increased the level of transcription activity of the HBV core promoter about 3.0-fold but had no effect on the other three promoters (Fig. 5A). As miR-1 does not bind to the HBV genome sequence directly, it may regulate specific transcription factors which bind to the HBV core promoter. Many nuclear receptors, like FXRA, HNF4A, PPARA, PPARG, RXRA, and LXR, were described as binding to the HBV core promoter and regulating HBV transcription and replication.15 A screening by real-time RT-PCR revealed an enhanced FXRA expression in HepG2.2.15 after miR-1 transfection (Fig. 5B). The expression of the other five receptors was not significantly changed. The up-regulation of FXRA expression was further verified by western blot (Fig. 5C). These results indicated that miR-1 may increase HBV transcription under the control of the HBV core promoter in an FXRA-dependent manner.

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Figure 5. MiR-1 transactivates HBV core promoter by augmenting FXRA expression. (A) Luciferase reporters containing HBV promoter regions pSP1, pSP2, pCP, and XP were cotransfected at a concentration of 100 ng/mL with 20 nM of miR-1 into HepG2.2.15 cells and assayed for luciferase activity at 48 hours. Luciferase activity was normalized against pGL3-basic control transfection. The relative luciferase expression was determined in miR-1 to miR-C transfected samples. (B) PPARA, PPARG, RXRA, FXRA, LXR, and HNF4A mRNA levels in HepG2.2.15 cells were assessed by real-time RT-PCR at day 4 following transfection with miR-1 and miR-C at 20 nM. Fold change was determined in miR-1 to miR-C transfected samples. (C) HepG2.2.15 cells were transfected with 20 nM of miR-1 or miR-C for 4 days and the FXRA protein expression levels were assessed by western blot with β-actin as the loading control. *P < 0.05.

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FXRA Blockage Attenuates miR-1-Mediated Enhancement of HBV Replication.

It has been reported that FXRA binds to two motifs on the HBV enhancer II and core promoter regions and increases the synthesis of HBV pregenomic RNA and RI.24 Thus, we asked whether miR-1 enhances HBV replication through FXRA. First, mutations in the FXRA binding motifs within the HBV core promoter abolished the miR-1 mediated activation of HBV core promoter (Fig. 6A). Further, the enhancement of HBV replication by miR-1 could be partially blocked by a natural FXRA antagonist GGS (Fig. 6B, lane 4). As GGS is also able to activate other steroid receptors,25 the role of FXRA was further confirmed by RNA silencing. An siRNA, siFXRA2, decreased the expression level of FXRA protein markedly, whereas another one, siFXRA1, was not effective (Fig. 6D). Cotransfection of miR-1 only with siFXRA2 blocked partially the up-regulation of HBV replication by miR-1 (Fig. 6C, lane 6). The nonsense siRNA control and siFXRA1 had no significant effect on HBV replication (Fig. 6C). Notably, both GGS and siFXRA2 also reduced the basal replication of HBV in the absence of ectopic miR-1 expression (Fig. 6B, lane 2, and 6C, lane 3). Thus, these data suggest that FXRA is involved in the action of miR-1 on HBV replication.

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Figure 6. FXRA blockage attenuates miR-1 mediated enhancement of HBV replication. (A) The reporter vectors pEN2/CP and pEN2/CP EmCm were cotransfected at a concentration of 100 ng/mL with 20 nM of miR-1 into HepG2.2.15 cells and assayed for luciferase activity at 48 hours. Luciferase activity was normalized against pGL3-basic as a control. (B) HepG2.2.15 cells were transfected with 20 nM of miR-1 or miR-C, and then treated with medium control or FXRA antagonist GGS (10 μM) for 4 days. HBV RI were detected by southern blot (middle panel) and quantified by real-time PCR (bottom panel) analysis. (C) HepG2.2.15 cells were cotransfected with 20 nM of miR-1 and 20 nM of FXRA specific siRNA (siFXRA1, siFXRA2) or siRNA-C for 4 days. The levels of HBV RI were determined as described above. (D) The knockdown of FXRA protein expression was examined by western blot after siFXRA1 and siFXRA2 transfection for 4 days with β-actin as the loading control. *P < 0.05.

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MiR-1 Inhibits Cell Proliferation and Arrests Cell Cycle by Targeting E2F5.

It has been described that miR-1 is able to target Foxp1, Met, and HDAC4 to regulate cell proliferation and cell cycle progression of HepG2 cells.21 Similarly, proliferation and DNA replication potential of HepG2.2.15 cells were decreased by miR-1 transfection (Supporting Information Fig. 5). Cell cycle distribution analysis showed that miR-1 transfection led to an increase of the cell population arrested at the G1 phase (Fig. 7A), even after treatment with the cell cycle inhibitor nocodazole blocking the cell cycle at the G2/M phase (Fig. 7A; Supporting Information Fig. 6). The most impressive evidence was obtained by synchronization of transfected cells at the G1 phase with aphidicolin. After withdrawal of aphidicolin, about 30% of miR-1-transfected cells remained in the G1 phase, whereas over 95% of control cells entered the S or G2/M phase, suggesting that G1/S cell cycle transition was slowed down by miR-1 (Fig. 7A; Supporting Information Fig. 6).

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Figure 7. MiR-1 arrests cell cycle by targeting E2F5. (A) HepG2.2.15 cells were transfected with 20 nM of miR-1 or miR-C for 48 hours, and then treated with nocodazole (100 nM), aphidicolin (4 μg/mL), or medium control for 24 hours. The distribution of cells in cell-cycle phases was assessed by flow cytometry using propidium iodide staining. The cell cycle analysis was performed in duplicate with two different batches of transfected cells. The percentage of cells in different phases of the cell cycle is shown. (B) The potential miR-1 binding site in 3′-UTR of E2F5, as predicted by TargetScan 5.1. (C) E2F5 3′-UTR containing the miR-1 target sequence was cloned into the 3′UTR of a luciferase reporter pMIR-REPORT. pmiR-E2F5-3UTR or control vector were cotransfected into HepG2 or Huh7 cells with miR-C, miR-1, or m-miR-1 and assayed for luciferase activity at 48 hours. Relative expression was determined in miR-1 and m-miR-1 to miR-con transfected samples. (D) HepG2.2.15 cells were transfected with 20 nM of miR-1 or miR-C for 4 days. E2F5 and phosphorylated-RB proteins levels were assessed by western blot with β-actin as the loading control. *P < 0.05.

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G1/S cell cycle transition is mainly controlled by the transcription complex that includes E2Fs, retinoblastoma (Rb), and HDACs.26 Silencing of HDAC4 by siRNA did not influence the cell cycle progression of HepG2.2.15 cells (data not shown). Different target prediction algorithms (MiRanda, TargetScan, and Pictar) identified E2F5 as a potential target of miR-1, with an evolutionarily conserved recognition site (nt 542-548) in its 3′UTR of its mRNA (Fig. 7B; Supporting Information Fig. 7). E2F5 represents a possible link to the blockage of G1/S cell cycle transition by miR-1, as E2F5 belongs to the E2F family of transcription factors which plays a crucial role in the cell cycle control.27 To verify whether E2F5 is a target of miR-1, pmiR-REPORT vector harboring E2F5 3′UTR sequence was cotransfected with miR-1 or miR-C into HepG2 or Huh7 cells, respectively. MiR-1, but not miR-C, specifically decreased the reporter gene luciferase expression of the E2F5 3′UTR reporter (Fig. 7C). Furthermore, the expression of E2F5 protein in HepG2.2.15 cells was determined after transfection with miR-1 or miR-C. Western blot analysis of whole cell extracts showed that the steady-state level of E2F5 was reduced by miR-1 in a dose-dependent manner (Fig. 7D). The sequential dephosphorylation of Rb under cell cycle arrest was also mediated by miR-1 transfection (Fig. 7D). Taken together, these results indicated that miR-1 targeted E2F5 to inhibit cell proliferation and arrested the cell cycle at the G1 phase.

MiR-1 Promotes a Differentiation Phenotype in HepG2.2.15 Cells.

HDAC inhibitors have been shown to be potent inducers of growth arrest and differentiation of transformed cells in vitro and in vivo.28 Previously, miR-1 was documented to promote cell differentiation by suppressing HDAC4 during muscle development.22 Thus, miR-1 may promote hepatoma cells to assume a more differentiated status. Therefore, the global cellular gene expression of HepG2.2.15 cells after miR-1 transfection was examined by microarray analysis. A cluster of liver-specific genes characteristic for differentiated hepatocytes were up-regulated more than 2.0-fold after miR-1 transfection, as compared with miR-C transfection (Fig. 8A, Supporting Information Fig. 8). The mRNA levels of four representative genes, apolipoprotein A1 (APOA-I), albumin (ALB), sulfotransferase 2A (Sult2A1), and fibrogen β (FGB) were further determined by real-time RT-PCR. Consistently, the mRNA levels of these genes were increased significantly after miR-1 transfection for 4 days (Fig. 8B). Increased ALB protein expression was additionally confirmed by western blot (Fig. 8C). Consistently, miR-1 mediated up-regulation of FXRA and down-regulation of E2F5 was also observed in microarray analysis (Fig. 8A). Thus, miR-1 is able to target multiple cellular genes to inhibit cell growth and promote cell differentiation of hepatoma cells, which is apparently beneficial for HBV replication.

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Figure 8. MiR-1 promotes a differentiation phenotype in HepG2.2.15 cells. (A) Microarray analysis of differentially regulated cellular genes in HepG2.2.15 cells after transfection with 20 nM of miR-1 or miR-C for 4 days. The gene expression profiles of miR-1 to miR-C transfected cells were compared and 50 differentially regulated genes with an increase over 2.0-fold or with a reduction ≥50% were selected. The represented heatmap for selected genes was generated by Spotfire according to the log ratio of fold changes. (B) After miR-1 and miR-C transfection, the expression levels of APOA-I, ALB, Sult2A1, and FGB were determined by real-time RT-PCR relative to 106 β-actin transcripts. (C) ALB protein levels were assessed by western blot with β-actin as the loading control. *P < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Recent data have shown that cellular miRNAs have the potential to directly boost viral replication in host cells, as shown for miR-122 and HCV.3 The data presented here, however, suggest that miRNAs may facilitate viral replication and persistence by modifying host gene expression. Our results demonstrate that miR-1 acts as a positive regulator of HBV replication and transcription through regulating the cellular gene expression.

MiR-1 has been shown to play an important role in many cellular and biological functions of the cardiovascular system. The presence of miR-1 in liver tissues and hepatoma cells has been reported.21, 29 By real-time PCR, we confirmed the expression of miR-1 in primary hepatocytes and hepatoma cell lines (Supporting Information Fig. 9), indicating that miR-1 may play a role in the regulation of hepatic gene expression, as well as HBV replication.

It is has been suggested that miRNAs may not only regulate gene expression at the posttranscriptional level, but that they are also capable of modifying chromatin.30 Because HBV covalently closed circular DNA (cccDNA) forms a viral minichromosome in infected hepatocytes as a template for the transcription of viral mRNAs, epigenetic modifications of the HBV cccDNA, such as the deacetylation of cccDNA-bound histones by HDACs, might regulate the transcription of viral chromatin and thereby viral replication.31 A recent study revealed that HBV replication is regulated by the acetylation status of H3/H4 histones bound to the HBV cccDNA, both in cell-based replication systems and in the liver of HBV chronically infected patients.23 We confirmed that HDAC4 expression is down-regulated by miR-1. Silencing of HDAC4 by siRNA or histone deacetylase inhibitors TSA treatment led to the enhancement of HBV replication. Our results are consistent with previous findings and suggest the significance of epigenetic modifications for HBV replication.

A direct link from miR-1 action to HBV replication is the regulation of HBV core promoter activity by augmenting FXRA expression. Several studies have described the essential role of FXRA/RXRA in the HBV life cycle in detail. FXRA forms a heterodimer with RXRA and binds to the regulatory sequences in the HBV core promoter. Activation of the FXR/RXR pathway by bile acids can enhance HBV transcription and replication.24, 32 Ectopic expression of FXRA/RXRA can establish HBV replication in nonhepatoma cells.15 In our study, FXRA was significantly up-regulated by miR-1. Furthermore, the FXRA antagonist GGS and FXRA-specific siRNA partially attenuated the miR-1 effect on HBV replication. Our results strongly suggested that FXRA may contribute to the modulation of HBV core promoter activity and subsequently to the level of viral replication by miR-1 expression. However, the mechanism of miR-1 regulation in FXRA expression remains to be clarified.

The ability of miRNAs to regulate the expression of multiple target genes implies the influence of miRNAs on global biological processes in cells. As shown previously, miR-1 expression was reduced in primary human hepatocellular carcinomas compared with matching normal liver tissue.21 In our study, we found that ectopic expression of miR-1 resulted in loss of cancer cell phenotype of hepatoma cells. MiR-1 may target E2F5 or other proliferation-related genes (like HDAC4, MET, and Foxp1)21 to slow down cell cycle progression and reduce cell proliferation. In addition, the analysis of the cellular gene expression profile revealed that overexpression of miR-1 resulted in up-regulation of multiple genes related to bile acid, cholesterol, amino acid, and glucose metabolism, reflecting a highly differentiated hepatocyte phenotype. Previous studies showed that the loss of differentiation status of hepatocytes may greatly reduce the ability of cells to support HBV replication.13 Li et al.33 showed that the replication of woodchuck hepatitis virus and viral antigen expression were gradually decreased early during preneoplastic cell lineages. In general, HBV replication is low or absent in HCC tissue which is associated with the dedifferentiation of hepatocytes. Our results suggested that ectopic expression of miRNAs in hepatoma cells may promote cell differentiation and restore, at least partially, the hepatocyte phenotype. Such cell culture systems will be beneficial for studies on HBV replication and drug screening because many cellular pathways are significantly modified in hepatoma cells in comparison with primary hepatocytes.

Recent research has emphasized that the dependence of the viral infection cycle on cellular factors is greater than previously anticipated. We hypothesize that HBV replication may be regulated by several miRNAs through redundant or nonredundant pathways. Further systematic testing of newly found miRNAs is warranted to find additional candidates. Identifying these host factors and characterizing their interactions with the viral and cellular components has the potential to reveal novel targets for specific antiviral strategies.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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