MicroRNA-196 represses Bach1 protein and hepatitis C virus gene expression in human hepatoma cells expressing hepatitis C viral proteins

Authors

  • Weihong Hou,

    Corresponding author
    1. Liver-Biliary-Pancreatic Center and the Liver, Digestive Diseases, and Metabolism Laboratory, Carolinas Medical Center, Charlotte, NC
    2. Department of Biology, the University of North Carolina at Charlotte, Charlotte, NC
    • Suite 210, Cannon Research Center, Carolinas Medical Center, 1000 Blythe Blvd., Charlotte, NC 28232-2861
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    • fax: 704-355-7648.

  • Qing Tian,

    1. Liver-Biliary-Pancreatic Center and the Liver, Digestive Diseases, and Metabolism Laboratory, Carolinas Medical Center, Charlotte, NC
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  • Jianyu Zheng,

    1. Liver-Biliary-Pancreatic Center and the Liver, Digestive Diseases, and Metabolism Laboratory, Carolinas Medical Center, Charlotte, NC
    2. Department of Biology, the University of North Carolina at Charlotte, Charlotte, NC
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  • Herbert L. Bonkovsky

    1. Liver-Biliary-Pancreatic Center and the Liver, Digestive Diseases, and Metabolism Laboratory, Carolinas Medical Center, Charlotte, NC
    2. Department of Biology, the University of North Carolina at Charlotte, Charlotte, NC
    3. Department of Medicine, the University of North Carolina at Chapel Hill, Chapel Hill, NC
    4. Department of Medicine, Microbial, & Structural Biology, the University of Connecticut Health Center, Farmington, CT
    5. Department of Molecular, Microbial, & Structural Biology, the University of Connecticut Health Center, Farmington, CT
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  • Potential conflict of interest: Dr. Bonkovsky is a consultant for Boehringer-Ingelheim. He received grants from merck and Verte. He is a consultant for and advises Novartis. He is a consultant for and is on the speakers' bureau of Ovation.

Abstract

Hepatitis C virus (HCV) directly induces oxidative stress and liver injury. Bach1, a basic leucine zipper mammalian transcriptional repressor, negatively regulates heme oxygenase 1 (HMOX1), a key cytoprotective enzyme that has antioxidant and anti-inflammatory activities. microRNAs (miRNAs) are small noncoding RNAs (≈22 nt) that are important regulators of gene expression. Whether and how miRNAs regulate Bach1 or HCV are largely unknown. The aims of this study were to determine whether miR-196 regulates Bach1, HMOX1, and/or HCV gene expression. HCV replicon cell lines (Con1 and 9–13) of the Con1 isolate and J6/JFH1-based HCV cell culture system were used in this study. The effects of miR-196 mimic on Bach1, HMOX1, and HCV RNA, and protein levels were measured by way of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. The Dual Glo Luciferase Assay System was used to determine reporter activities. miR-196 mimic significantly down-regulated Bach1 and up-regulated HMOX1 gene expression and inhibited HCV expression. Dual luciferase reporter assays demonstrated that transfection of miR-196 mimic resulted in a significant decrease in Bach1 3′-untranslated region (UTR)–dependent luciferase activity but not in mutant Bach1 3′-UTR–dependent luciferase activity. Moreover, there was no detectable effect of mutant miR-196 on Bach1 3′-UTR–dependent luciferase activity. Conclusion: miR-196 directly acts on the 3′-UTR of Bach1 messenger RNA and translationally represses the expression of this protein, and up-regulates HMOX1. miR-196 also inhibits HCV expression in HCV replicon cell lines (genotype 1b) and in J6/JFH1 (genotype 2a) HCV cell culture system. Thus, miR-196 plays a role in both HMOX1/Bach1 expression and the regulation of HCV expression in human hepatocytes. Overexpression of miR-196 holds promise as a potential novel strategy to prevent or ameliorate hepatitis C infection, and to protect against liver injury in chronic HCV infection. (HEPATOLOGY 2010.)

Hepatitis C virus (HCV) infection is a worldwide health problem. The current standard therapy for chronic hepatitis C is a combination of pegylated interferon and ribavirin, but only ≈50% of patients respond to such treatment, which is expensive, prolonged, and attended by numerous unpleasant side effects.1, 2 There is a continuing need to develop new antiviral therapies. The molecular mechanisms underlying HCV-associated liver injury remain imperfectly understood. However, studies have indicated that HCV directly induces oxidative stress in hepatocytes through multiple mechanisms that include chronic inflammation, increases in iron, and liver injury. Therefore oxidative stress has emerged as an important pathogenetic mechanism in chronic hepatitis C,3–5 and antioxidant and cytoprotective therapy has been proposed to treat hepatitis C.

Heme oxygenase 1 (HMOX1) is a key cytoprotective enzyme, catalyzing heme degradation and generating ferrous iron, carbon monoxide, and biliverdin, the latter two of which have antioxidant and anti-inflammatory activities in vivo.6–8 Bach1, a basic leucine zipper mammalian transcriptional repressor of HMOX1, negatively regulates HMOX1 gene expression. Bach1 forms antagonizing heterodimers with the Maf-related oncogene family. These heterodimers bind to Maf recognition elements and suppress expression of genes [e.g., HMOX1 and NAD(P)H:quinone oxidoreductase].9–12

microRNAs (miRNAs) are a class of small noncoding RNAs (≈22 nt) that regulate gene expression primarily through translational repression.13, 14 The exact mechanism by which target genes are down-regulated remains unclear. Sequence complementarity in the 6- to 8-bp seed regions at the end of miRNA–messenger RNA (mRNA) heteroduplexes seem to determine the specificity of miRNA–target RNA interactions.15 miR-196 was first recognized to have extensive and evolutionarily conserved complementarity to homeobox clusters, groups of related transcription factor genes crucial for numerous developmental programs in animals, and to regulate homeobox gene expression.16, 17 A recent report indicated one perfect match between miR-196 and nonstructural (NS) 5A coding region of the HCV JFH1 genome (genotype 2a), and interferon-β treatment resulted in significant induction of miR-196 in the human hepatoma cell line Huh-7 and in primary murine hepatocytes.18 A search of the TargetScan 4.2 database revealed that at least two miR-196 seed match sites occur in the 3′-untranslated region (UTR) of Bach1 mRNA. This led us to propose that miR-196 is an miRNA that targets the HCV genome and the 3′-UTR of Bach1 mRNA, the latter leading to up-regulation of the HMOX1 gene. These dual effects are analogous to effects of miR-122, recently reported from our laboratory.19

In this study, we experimentally validated the computational prediction of miR-196 binding in the 3′-UTR of Bach 1 mRNA by luciferase reporter assay. miR-196 mimic significantly down-regulated Bach1 and up-regulated HMOX1 gene expression, and inhibited HCV expression. These results suggest that up-regulation of miR-196 may be an additional new therapeutic approach to prevent or ameliorate hepatitis C infection, and to protect against liver injury in chronic HCV infection. The findings indicate that miR-196 plays a role in the regulation of HMOX1/Bach1 expression and HCV replication in hepatocytes. They also add to the growing evidence that up-regulation of HMOX1 may be beneficial in HCV infection.5, 19, 20

Abbreviations

GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCV, hepatitis C virus; HMOX, heme oxygenase; miRNA, microRNA; MMNC, miRNA mimic negative control; mRNA, messenger RNA; Mut, mutant ; NS, nonstructural; qRT-PCR, quantitative real-time polymerase chain reaction; siRNA, small interfering RNA; UTR, untranslated region; WT, wild-type.

Materials and Methods

Cell Culture.

R. Bartenschlager (University of Heidelberg, Heidelberg, Germany) kindly provided 9–13 cells. These cells harbor a replicating HCV NS region with the use of the NS3–NS5B gene regions from the Con1 isolate.21 Huh-7.5 and Con1 subgenomic genotype 1b HCV replicon cell lines were from Apath LLC (St. Louis, MO). Huh-7.5 is a highly permissive, interferon-α–cured Huh-7 human hepatocellular carcinoma cell line derivative. The Con1 cell line is a Huh-7.5 cell population containing the full-length HCV genotype 1b replicon. The 9–13, Huh-7.5, and Con1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and selection antibiotic 500 μg/mL G418 for 9–13 cells, or 750 μg/mL G418 for Con1 cells.

miRNAs, Small Interfering iRNAs, and Reporter Constructs.

The miRIDIAN miRNA mimics for has-miR-196, has-miR-16, customized mutant has-miR-196, miRNA mimic negative control (MMNC), and Bach1–small interfering RNA (siRNA) were obtained from Dharmacon (Lafayette, CO). pRL-TK vector was obtained from Promega. The pRL-TK reporter vector contains a complementary DNA (Rluc) encoding Renilla luciferase as an internal control reporter. pGL3-Bach1 luciferase reporter construct, containing a 1,837-bp fragment of Bach1 3′-UTR, was a kind gift of Dr. Rolf Renne (University of Florida, Gainesville, FL).22 Mutant pGL3-Bach1 was generated by GENEWIZ, Inc. (South Plainfield, NJ). pLSV40-Rluc and pLSV40-GL3/Bach1 reporter vectors were kindly provided by B. R. Cullen (Duke University, Durham, NC). pLSV40-Rluc contains a complementary DNA (Rluc) encoding Renilla luciferase as an internal control reporter and pLSV40-GL3/Bach1 firefly luciferase reporter construct contains the full-length 3′-UTR of Bach1 mRNA.23 Constructs were confirmed by way of restriction enzyme digestion and sequencing.

Transfection and Luciferase Activity Assays.

Transfection of miR-196 mimic or Bach-siRNA was performed as described.24 Cotransfection of miRNA mimics and reporters were performed using Lipofectamine 2000 from Invitrogen (Carlsbad, CA) according to the manufacturer's protocol. Briefly, cells were cotransfected with 0.4 μg/mL of pGL3-Bach1 or mutant pGL3-Bach, with 0.4 μg/mL of pRL-TK, and with 10–50 nM tested miRNAs. 24 to 48 hours after transfection, cells were harvested and lysed, and the luciferase reporter activities were measured using the Dual-Glo Luciferase Assay System. Firefly luciferase activity was normalized to Renilla luciferase activity and total protein, determined using the bicinchoninic acid protein assay kit. For ease of comparison, values for cells with pGL3-Bach1 and pRL-TK transfection were set equal to 1.

In Vitro Transcription, Transfection, and Infection.

The HCV infectious clone pJ6/JFH1, the full-length chimeric genome with the core-NS2 regions from the infectious J6 (genotype 2a) and NS3-NS5B regions from the infectious JFH1 (genotype 2a), was generously provided by C. M. Rice (the Rockefeller University, New York, NY). The production of J6/JFH1-based cell culture–generated HCV has been reported.25 In brief, the pJ6/JFH1 plasmid was linearized with XbaI and purified by ethanol precipitation, digestion with proteinase K, and phenol-chloroform extraction. The linearized plasmid was used as a template for in vitro transcription using the MEGAscript T7 kit (Ambion, Austin, TX). For HCV RNA transfection, Huh-7.5 cells were plated in 24-well plates 1 day prior to transfection and transfected at 70%–80% confluence. Cells were transfected at an RNA/Lipofectamine ratio of 1:2 by using 2 μg/well of HCV RNA and 4 μL/well Lipofectamine 2000 for 48 hours. To infect naïve Huh-7.5 cells, cell culture supernatants from the cells transfected with HCV RNA for 48 hours were collected and filtered through a 0.20 μm filter, and added to cultures of naïve Huh-7.5 cells.

The pFK-Con1 construct (genotype 1b)21 was a kind gift of Dr. R. Bartenschlager (University of Heidelberg, Heidelberg, Germany). Mutant pFK-Con1 (pFK-Con1-Mut) with mutations in four nucleotides in the putative binding sites for miR-196 was generated by GENEWIZ, Inc. (South Plainfield, NJ) and confirmed by sequencing. pFK-Con1–wild-type (WT) and pFK-Con1-Mut were linearized with AseI and ScaI. In vitro transcription and transfection were performed as described.

Western Blotting.

Western blotting was performed using the standard protocols of our laboratory as reported.10 The method is described in detail in the Supporting Materials and Methods.

qRT-PCR.

Total RNA from tested cells was extracted, and complementary DNA was synthesized.10 qRT-PCR was performed using primers as reported19, 26 and as described in detail in the Supporting Material and Methods.

Statistical Analysis.

Initial analysis showed that results were normally distributed. Therefore, parametric statistical procedures were used. The Student t test (for comparison of two conditions) or analysis of variance (for comparisons among more than two) was used to analyze the differences among means. Values of P < 0.05 were considered statistically significant. Experiments were repeated at least three times with similar results. All experiments included at least triplicate samples for each treatment group. Representative results from single experiments are presented. Statistical analyses were performed with JMP 4.0.4 software from SAS Institute (Cary, NC).

Results

Analysis In Silico Predicts Two Seed Region Matches of miR-196 and the 3′-UTR of Bach1 mRNA.

An online search of the TargetScan 4.2 database (http://www.targetscan.org/vert_42/) demonstrated that at least two putative miR-196 seed match sites were harbored in the 3′-UTR of Bach1 mRNA. As shown in Fig. 1A,B, one of the predicted binding sites (2,280–2,286 nt) was highly conserved in human, mouse, rat, chicken, and dog, whereas the other putative site (2,161–2,166 nt) was poorly conserved across species. No predicted miR-196 binding sites were found in the nuclear regulatory factor erythroid 2–related factor 2 and HMOX1 gene, and no putative miR-196 binding sites were found in the coding region of Bach1 gene (data not shown).

Figure 1.

In silico prediction of putative binding sites for miR-196 in the 3′-UTR of Bach1. (A) Schematic of seed region match between miR-196 and the first putative Bach1 3′-UTR site targeted. The first site is highly conserved in the human, mouse, rat, chicken and dog. (B) Schematic of second seed region match between miR-196 and the putative Bach1 3′-UTR site targeted. The second site is poorly conserved in the human, mouse, rat, dog, and pig.

miR-196 Down-Regulates the Transcriptional Repressor Bach1 and Up-Regulates HMOX1 in 9-13 Cells Expressing HCV NS Proteins.

To experimentally verify that the putative miR-196 binding sites are functional, we transfected 9–13 cells with miR-196–specific mimic and measured Bach1 protein and mRNA levels by way of Western blotting and qRT-PCR, respectively. 9–13 cells transfected with miR-196 mimic showed a significant reduction in the expression of Bach1 protein levels (≈55% after 24 hours' transfection and ≈64% after 48 hours' transfection) compared with MMNC, whereas no effects on Bach1 protein levels were detectable in cells transfected with miRNA mimic negative control compared with mock transfection (Fig. 2A). However, no significant effect of miR-196 on Bach1 mRNA levels was observed in 9–13 cells (Fig. 2B). These results demonstrate that the regulation of miR-196 on Bach1 occurs at a translational level in human hepatoma 9–13 cells.

Figure 2.

miR-196 mimic down-regulates Bach1 protein levels and up-regulates expression of the HMOX1 gene in 9–13 cells. 9–13 cells were transfected with 50 nM miR-196 mimic or MMNC by Lipofectamine 2000 as indicated. 24 to 48 hours after transfection, the cells were harvested. Bach1 protein levels were assessed by way of Western blotting with anti-Bach1 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific antibodies. The levels of Bach1 mRNA were quantified using qRT-PCR as described in Materials and Methods. The amounts of Bach1 protein and mRNA were normalized to GAPDH which did not vary with treatment. Values for cells with a mock transfection were set equal to 1. The levels of HMOX1 mRNA were quantified by qRT-PCR. The amounts of HMOX1 mRNA levels were normalized to GAPDH which did not vary with transfection. Values for cells with mimic negative control transfection were set equal to 1. Data are presented as the mean ± standard error (n = 3). *P < 0.05 versus negative control. (A) miR-196 mimic down-regulated Bach1 protein levels. (B) The levels of Bach1 mRNA were not altered with miR-196 mimic transfection. (C) miR-196 mimic up-regulated HMOX1 mRNA levels. (D) miR-196 did not alter Cullin 3 mRNA levels.

Bach1 is a well-established transcriptional repressor of the HMOX1 gene10, 11; therefore, we next determined whether down-regulation of Bach1 protein by miR-196 could increase HMOX1 gene expression. 9–13 cells were transfected with miR-196 mimic or miRNA mimic negative control for 48 hours, after which the levels of HMOX1 and Cullin 3 (Cul 3, nonspecific gene control) mRNA were quantified by way of qRT-PCR. As expected, miR-196 mimic significantly up-regulated HMOX1 mRNA levels by ≈2.4-fold (Fig. 2C), but not Cul 3 mRNA levels (Fig. 2D) compared with the same amount of miRNA mimic negative control.

miR-196 Directly Interacts with the 3′-UTR of Bach1 mRNA in 9–13 Cells Expressing HCV NS Proteins.

To further establish that miR-196 targets the 3′-UTR of Bach1 mRNA, which contains two predicted seed match sites for miR-196 (Fig. 3A), (rather than exerting a less direct and specific regulation), a reporter construct, which we called pGL3-Bach1, with Bach1 3′-UTR downstream of the firefly luciferase (f-luc) open reading frame (Fig. 3B), was used. 9-13 cells were cotransfected with pGL3-Bach1 (f-luc), pRL-TK (renilla, to normalize for transfection efficiencies), and miRNA-negative controls, miR-196 mimic, or miR-16 (a negative miR with no predicted binding sites in the 3′-UTR of Bach1 mRNA). Forty-eight hours after transfection, the luciferase reporter activity was assayed. miR-196 mimic transfection significantly decreased reporter activity by ≈53%, whereas miRNA mimic negative control and miR-16 mimic had no effect on reporter luciferase activity (Fig. 3C). A similar effect was observed when we cotransfected 9-13 cells with a reporter construct pLSV40-GL3/Bach1 containing the full length 3′-UTR of Bach1 mRNA and miR-196 mimic. Specifically, 50 nM of miR-196 mimic decreased the firefly luciferase activity by ≈59% (Supporting Fig. 1B), whereas MMNC had no significant effect on reporter luciferase activity (Supporting Fig. 1C).

Figure 3.

miR-196 mimic represses the expression of a luciferase reporter containing Bach1 3′-UTR. (A) The Bach1 3′-UTR contains two seed match sites (highlighted) for miR-196. (B) Schematic representation of pGL3-Bach1, the firefly luciferase (f-luc) reporter construct used. (C) Inhibition of the f-luc activities of pGL3-Bach1 3′-UTR reporter by miR-196 mimic. 9-13 cells were cotransfected with 0.4 μg/mL of pGL3-Bach1, 0.4 μg/mL of pRL-TK (renilla) and with 50 nM miR-196 mimic, miR-16 mimic, or MMNC by Lipofectamine 2000 as indicated. Forty-eight hours after transfection, the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega. Firefly luciferase activities were normalized to renilla luciferase activities and total protein, as indicated in Materials and Methods. Values for cells with a mock transfection were set equal to 1. Data are presented as the mean ± standard error (n = 3). *P < 0.05 versus negative control.

Next, we cotransfected 9-13 cells with luciferase reporter construct containing a 4-nt mutant Bach1 3′-UTR, which we called pGL3-Bach1-Mut (Fig. 4A), and with pRL-TK, and with miR-196 mimic or miR-155 mimic22 (a negative miR, with no changes of predicted miR-155 binding sites in pGL3-Bach1-WT and pGL3-Bach1-Mut), and assayed the luciferase reporter activity. miR-196 mimic transfection significantly decreased luciferase activity in a dose-dependent fashion, whereas miR-196 did not change reporter activity in cells transfected with the reporter construct containing mutant binding sites for miR-196 (Fig. 4B). miR-155 significantly decreased reporter activity in both pGL3-Bach1-WT and pGL3-Bach1-Mut (Fig. 4C). These results demonstrate that miR-196 directly regulates Bach1 gene expression and miR-196 mediates down-regulation of Bach1 though the 3′-UTR of Bach1 mRNA.

Figure 4.

miR-196 does not inhibit luciferase activity of reporter with mutant Bach1 3′-UTR. (A) Four nucleotides in two seed match sites of Bach1 3′-UTR were replaced. (B) miR-196 mimic decreased the f-luc activity of pGL3-Bach1-WT but not pGL-Bach1-Mut reporter. 9-13 cells were cotransfected with 0.4 μg/mL of mutant pGL3-Bach1 or 0.4 μg/mL of pGL3-Bach1, with 0.4 μg/mL of pRL-TK (renilla), and with miR-196 mimic (10-50 nM) as shown in Materials and Methods. Forty-eight hours after transfection, the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega, and firefly luciferase activities were normalized to renilla luciferase activities and total protein. (C) miR-155 mimic decreased the f-luc activities of both pGL3-Bach1-WT and pGL-Bach1-Mut reporter. 9-13 cells were cotransfected with 0.4 μg/mL of mutant pGL3-Bach1 or 0.4 μg/mL of pGL3-Bach1, with 0.4 μg/mL of pRL-TK (renilla), and with miR-155 mimic (10-50 nM). Forty-eight hours after transfection, the luciferase reporter activities were assayed as described in Materials and Methods. Values for cells with a mock transfection were set equal to 1. Data are presented as the mean ± standard error (n = 3). *P < 0.05 versus negative control.

To further establish the direct interaction between miR-196 and Bach1–3′-UTR, we created mutant miR-196 (Fig. 5A) in which seed match sites for the 3′-UTR of Bach1 mRNA were abolished. Cells were cotransfected with pGL3-Bach1-WT, and with pRL-TK, and with increasing concentrations of miRNA mimic negative control, miR-196 mimic, or mutant miR-196 mimic, and luciferase reporter activity was assayed. As anticipated, miR-196 resulted in a significant reduction in luciferase activity in a dose-dependent fashion, which was consistent with our previous observations, whereas miRNA negative controls and mutant miR-196 did not affect luciferase activity (Fig. 5B), further indicating the direct interaction between miR-196 and the 3′-UTR of Bach1 mRNA.

Figure 5.

Mutant miR-196 mimic does not alter the luciferase activity of Bach1 3′-UTR reporter but inhibits the luciferase activity of mutant Bach1 3′-UTR reporter. (A) Four-nucleotide mutations were introduced to the seed match sites of miR-196. (B) Effect of mutant miR-196 on Bach1 3′-UTR reporter activity. 9-13 cells were cotransfected with 0.4 μg/mL of pGL3-Bach1, 0.4 μg/mL of pRL-TK, and with 0, 10, 20, or 50 nM mimic negative control, miR-196 mimic, or mutant miR-196 for 48 hours. Firefly and renilla luciferase activities were measured using Dual Luciferase Assay System (Promega). Firefly luciferase activities were normalized to renilla luciferase activities and total protein as described in Materials and Methods. Values for cells without miRNA transfection were set equal to 1. (C) Restoration of seed match between mutant miR-196 and mutant Bach1 3′-UTR are shown. (D) Inhibition of mutant Bach1 3′-UTR reporter activity by mutant miR-196 mimic. 9-13 cells were cotransfected with 0.4 μg/mL of mutant reporter (pGL3-Bach1-Mut), 0.4 μg/mL of pRL-TK, and with 0, 10, 20, or 50 nM Mut miR-196 or WT miR-196 mimic for 48 hours. Firefly and renilla luciferase activities were measured. Firefly luciferase activities were normalized to renilla luciferase activities and total protein. Values for cells without miRNA transfection were set equal to 1. Data are presented as the mean ± standard error (n = 3). *P < 0.05 versus negative control.

We predicted that mutant miR-196 (miR-196-Mut) containing base complementarity with mutant pGL3-Bach1 (pGL3-Bach1-Mut) should restore its effect on mutant reporter (Bach1-3′-UTR-Mut) activity, because they again match perfectly in their seed regions (Fig. 5C). As we predicted, mutant miR-196 mimic significantly inhibited luciferase activity in cells transfected with pGL3-Bach1-Mut, which was mutated to fit mutant miR-196, whereas no significant effects of miR-196-WT on mutant reporter (pGL3-Bach1-Mut) luciferase activity were observed, further establishing the direct interaction between miR-196 and the 3′-UTR of Bach1 mRNA (Fig. 5D).

miR-196 Represses HCV RNA and Protein Expression in HCV Replicon Cells.

To determine whether miR-196 represses HCV mRNA and protein expression, HCV replicon cell lines Con1 and 9-13 cells were transfected with miR-196 mimic or miRNA mimic negative control. As shown in Fig. 6A,B, 50 nM of miR-196 mimic resulted in a significant reduction of HCV replication by ≈50% in Con1 cells and NS5A protein levels by ≈52%, compared with the same concentration of miRNA mimic negative control. When Bach1 gene expression was silenced by Bach1-siRNA (Fig. 6C), the levels of HCV replication were significantly decreased by ≈40% (Fig. 6D).

Figure 6.

miR-196 mimic represses HCV expression in HCV replicon cells. Cells were transfected for 24 to 48 hours with 10-50 nM miR-196 mimic or Bach1-siRNA by Lipofectamine 2000, after which cells were harvested and total RNA and protein were extracted. The levels of HCV core and NS5A and host cell GAPDH mRNA and protein levels were measured by way of qRT-PCR and Western blotting as described in Materials and Methods. Data are presented as the mean ± standard error (n = 3). *P < 0.05 versus negative control. (A) Down-regulation of HCV core and NS5A mRNA expression by miR-196 mimic in Con1 cells. (B) Down-regulation of HCV NS5A protein expression by miR-196 mimic in 9-13 cells. (C) Silencing Bach1 gene expression by Bach1-siRNA in Con1 cells. (D) Down-regulation of HCV core and NS5A mRNA expression by Bach1-siRNA in Con1 cells.

Comparative Studies of miR-196 on HCV Expression in Con1-WT– Versus Con1-Mut–Transfected Huh-7.5 Cells.

Analysis in silico predicts two 6-nt match sites within HCV Con1 genome to a region of miR-196 (nucleotides 2-7) (Fig. 7A), which are not considered as perfect matches of a 7-nt match to the seed region of the miRNA (nucleotides 2-8), and different from the perfect seed match between miR-196 and HCV JFH1 genome.18 However, there is a possibility that two 6-nt complementary matches could lead to an effect. Therefore, we investigated whether the inhibition of HCV expression by miR-196 is the result of an effect through Bach1 and HMOX1 or by directly targeting the HCV genome, or both. We introduced 4-nt mutants to generate mutant pFK-Con1-Mut, in which two match sites for the HCV Con1 genome were abolished (Fig. 7A). 50 nM of miR-196 transfection still resulted in a significant reduction of HCV core and NS5A expression in Huh-7.5 cells transfected with Con1-Mut RNA, as observed in Huh-7.5 cells transfected with Con1-WT RNA (Fig. 7B,C), but the effect of miR-196 on HCV core and NS5A in Con1-Mut–transfected cells was slightly less than that in Con-WT–transfected cell (Fig. 7D). These findings, together with data shown in Fig. 6C,D, suggested that the inhibition of HCV expression by miR-196 is the result of an effect through Bach1 as well as targeting the HCV Con1 genome, and the indirect effect of miR-196 on HCV expression though Bach1 and HMOX1 is a major contribution to inhibition of HCV expression.

Figure 7.

Effect of miR-196 on HCV expression in Huh-7.5 cells transfected with Con1-WT or Con1-Mut RNA. Huh-7.5 cells were transfected with 2 μg/well of HCV Con1-WT or Con-Mut RNA by Lipofectamine 2000. After 24 hours, cells were transfected with 50 nM of miR-196 mimic or MMNC as control. After 48 hours, cells were harvested and total proteins were extracted. HCV core, NS5A, and GAPDH protein levels were measured by way of Western blotting. Data are presented as the mean ± standard error (n = 3). *P < 0.05 versus negative control. (A) In silico prediction of putative binding sites for miR-196 in HCV genotype 1 Con1 genome and the 4-nt mutation introduced to the binding sites of miR-196. (B) Effects of miR-196 on HCV core and NS5A protein levels in Con1-WT–transfected Huh-7.5 cells. (C) Effects of miR-196 on HCV core and NS5A protein levels in Con1-Mut–transfected Huh-7.5 cells. (D) Comparison of the effects in Con1-WT– versus Con1-Mut–transfected Huh-7.5 cells.

miR-196 Inhibits HCV Expression in the HCV JFH1-Based Cell Culture System.

As reported recently, a perfect match for miR-196 was found in the coding region of the HCV NS5A gene in the HCV JFH1 genome.18 As expected, we observed a similar down-regulatory effect of miR-196 on HCV expression in the HCV J6/JFH1 cell culture system. 50 nM of miR-196 led to a significant decrease of HCV J6/JFH1 RNA by nearly 70% in J6/JFH1 transfected Huh-7.5 cells (Fig. 8A), ≈50% in J6/JFH1 infected Huh-7.5 cells (Fig. 8B) and ≈60% reduction of HCV NS3 protein in J6/JFH1 infected Huh-7.5 cells (Fig. 8C). These results were consistent with previous observations in the JFH1 cell culture system.18

Figure 8.

miR-196 mimic inhibits HCV expression in the HCV J6/JFH1-based cell culture system. Huh-7.5 cells were transfected with 2 μg/well of HCV J6/JFH1 RNA by Lipofectamine 2000. After 48 hours, cells were transfected with miR-196 mimic or MMNC. For HCV infection, naïve Huh-7.5 were cultured with 1 mL of cell culture supernatants harvested from J6/JFH1-transfected cells as described in Materials and Methods. After 48 hours of exposure to the supernatants, cells were transfected with miR-196 mimic, or MMNC for 48 hours. Cells were harvested and total RNA and proteins were extracted. HCV RNA was quantified by way of qRT-PCR, and HCV NS3 and GAPDH protein levels were measured by way of Western blotting. Data are presented as the mean ± standard error (n = 3). *P < 0.05 versus negative control. (A) Down-regulation of HCV J6/JFH1 RNA levels by miR-196 mimic in Huh-7.5 cells transfected with J6/JFH1 RNA. (B) Down-regulation of HCV J6/JFH1 RNA and (C) protein levels by miR-196 mimic in Huh-7.5 cells infected with J6/JFH1 hepatitis C virus secreted into the culture supernatant.

Discussion

The major novel findings of this work are that (1) miR-196 has functional binding sites in the 3′-UTR of Bach1 mRNA and (2) down-regulation of Bach1 by either miR-196 or Bach1-siRNA represses HCV expression. Functionality of miR-196 in regulation of Bach1 is demonstrated by several lines of evidence, including initial in silico analysis (Fig. 1); by expected effects of miR-196 mimics (Figs. 2); and by the expected effects of miR-196 mimics on luciferase reporter constructs containing the WT and Mut Bach1 3′-UTR (Figs. 3–5; Supporting Fig. 1). In addition, we demonstrate that down-regulation of Bach1 by either miR-196 or Bach1-siRNA leads to the up-regulation of the HMOX1 gene (Fig. 2C,D) and to down-regulation of HCV expression in replicon cells (the genotype 1b Con1 strain) (Fig. 6) and the HCV J6/JFH1-generated cell culture system (Fig. 8). These results suggest that overexpression or up-regulation of miR-196 represents a potential new additional therapeutic strategy for chronic HCV infection, as well as other conditions in which up-regulation of HMOX1 may be desirable.

Based on a large number of experimental and clinical studies performed during the past several years, it is now generally accepted that HCV infection produces an increase in oxidative stress in infected hepatocytes. One important mediator of such increased oxidative stress is the HCV core protein.27, 28 In parallel with these observations are a series of observations in numerous systems, including experimental systems with expression of HCV, showing that HMOX1 helps to protect numerous cells and tissues against the potentially damaging effects of excess oxidative stress. These actions are based on the ability of HMOX1 to decrease free or loosely bound heme, which can act as a potent prooxidant, and to increase production of carbon monoxide, biliverdin, and bilirubin, which have potent antioxidant and anti-inflammatory and antifibrogenic effects.6-8, 29, 30 HMOX1 has also emerged as an important antiapoptotic enzyme.31 Overexpression or induction of HMOX1 suppresses HCV replication and increases resistance of hepatocytes to oxidant injury.19, 20

Regulation of expression of the HMOX1 gene is complex. However, we and others have shown that among the important sites for regulation are a series of expanded AP-1 sites, also called antioxidant responsive elements,31 Maf protein responsive elements, and metalloporphyrin-responsive elements in the 5′-UTR of HMOX genes, across many species.32-36 Bach1 plays a key role in tonic repression of expression of the HMOX1 gene. It does so by forming heterodimers with small Maf proteins and blocking transcriptional activation of the gene. Bach1 contains several consensus binding sites (all containing CP motifs), which when they bind heme, lead to a change in conformation of the protein with marked reduction in affinity for Maf proteins and subsequent derepression and increase in activity of HMOX1 gene expression.9, 10, 12

In view of the above, it is not surprising that HMOX1 activity might be increased in HCV infection, and, indeed, we and others have shown this to be the case.5 Nevertheless, in some other experimental systems and also in some clinical studies, a decrease in expression of HMOX1 has been observed in the setting of chronic hepatitis C.37, 38 These findings suggest that patients with genetic or other factors that lead to lower levels of HMOX1 gene expression may be at increased risk for development of chronic hepatitis C infection after acute HCV exposure and/or with greater risks of development of more rapidly progressive liver disease due to HCV infection. In this regard, there are at least two known genetic factors that influence levels of expression of HMOX1—namely, the length of GT repeats in the 5'-UTR and the presence of a single-nucleotide polymorphism at position -413 (A/T, rs2071746) in the HMOX1 promoter region.39, 40

The discovery of miRNAs in Caenorhabditis elegans ushered in a new and exciting area of study. The numbers of miRNAs continues to grow, and additional mRNAs and candidate genes regulated by them continue to be identified. With respect to the liver, miR-122 was identified as the most abundant miRNA expressed in hepatocytes (accounting for ≈70% of total miRNAs) and shown to have major effects on several enzymes of cholesterol metabolism.41, 42 Unexpectedly, miR-122 was also shown to be required for HCV expression,19, 43 at least in cell culture systems. More recent work has shown that the effects of miR-122 depend upon the context and location of its cognate seed sequence binding sites. The sites in the 5′-UTR are mostly associated with up-regulation of expression, whereas those in the 3′-UTR are mostly associated with repression of expression.44 The present study adds miR-196 as a down-regulator of HCV expression (Figs. 6 and 8) and an attractive candidate as new therapeutic agents for chronic HCV infection.

Our study has limitations. Effects of miR-196 on, Bach1, HMOX1, and HCV thus far have been shown only in cell culture models, and the suppression of HCV expression has been moderate, not extremely high. The field of HCV research has been stymied by the lack of simple and robust animal models. Among nonhuman species, only chimpanzees have thus far been capable of being infected with HCV, and the disease in them is generally relatively mild. They are also extraordinarily difficult and expensive to maintain. Recently, murine models have been developed, based on immunodeficient animals into which human hepatocytes are implanted without rejection and then infected with the hepatitis C virus.45, 46 Another recent model has been able to establish this in non-immunodeficient mice in which the host animal hepatocytes undergo necrosis and apoptosis and can be rescued with human hepatocytes.47 Thus, overexpression of a combination of miR-196 and other selected miRNAs in order to decrease the viral output further in cell cultures and murine models are currently under study in our laboratory.

In conclusion, we demonstrate functional miR-196 binding sites in the 3′-UTR of Bach1, which lead to down-regulation of Bach1 gene expression, up-regulation of HMOX1 gene expression, and down-regulation of HCV expression. These findings add to the growing panoply of miRNAs that influence expression of genes and proteins of the hepatitis C virus and of HMOX1, a key cytoprotective enzyme. They suggest potential new additional therapies for chronic HCV infection and, perhaps, for other diseases characterized by increased oxidative stress.

Acknowledgements

We thank Dr. Rolf Renne (University of Florida, Gainesville, FL) for the generous gift of luciferase reporter construct pGL3-Bach1 and Dr. Bryan R. Cullen (Duke University, Durham, NC) for providing pLSV40-Rluc, pLSV40-GL3, and pLSV40-GL3/Bach1 reporter vectors. We are grateful to Dr. Ralf Bartenschlager (University of Heidelberg, Heidelberg, Germany) for kindly supplying the HCV 9-13 cells and pFK-Con1 plasmid, and Dr. Charles M. Rice (the Rockefeller University, New York, NY) for providing the J6/JFH1 molecular clone.

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