Journal of Viral Hepatitis, 2014, 21, 121–128
doi:10.1111/jvh.12131
MicroRNA-130a inhibits HCV replication by restoring the innate immune response S. Li,1,* X. Duan,1,* Y. Li,1 B. Liu,1 I. McGilvray1,2 and L. Chen1,2
1
The Institute of Blood Transfusion,
2
Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China; and Toronto General Research Institute, University of Toronto, Toronto, ON, Canada Received March 2013; accepted for publication May 2013
SUMMARY. Hepatitis C virus (HCV) infection is a major
cause of chronic hepatitis and hepatocellular carcinoma. Currently pegylated interferon (IFN) combined with ribavirin remains the best therapeutic approach, although patients infected with HCV genotype I may benefit from adding protease inhibitors as ‘triple therapy’. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression and have recently been shown to play an important role in human innate immune response and as an antiviral in chimpanzees. We studied the effect of miR-130a on the HCV replication. We found that miR130a significantly inhibits HCV replication in both HCV replicon and J6-/JFH1-infected cells. Over expression of
INTRODUCTION Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) [1]. There are about 170 million people infected with HCV worldwide. Although direct-acting antivirals (DAAs) targeting HCV polymerases or proteases are now available, interferon alpha (IFN-a) combined with ribavirin remains the standard care for treatment for chronic HCV infection in most developing countries [2].There is good reason to think that IFN-a will remain part of the treatment regimen for many years to come. HCV is an extremely mutagenic virus, and resistance has been described to every DAA to date [3–5]. Although IFN-a-free combinations of DAAs have been attempted Abbreviations: DAA, direct-acting antivirals; HCC, hepatocellular carcinoma; ISGs, interferon-stimulated genes; LNA, locked nucleic acid; MMNC, miRNA mimic negative control; PFV, primate foamy virus; UTR, untranslated region. Correspondence: Dr. Limin Chen, The Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, Sichuan, China. E-mail: [email protected] *These authors contributed equally to this study.
© 2013 John Wiley & Sons Ltd
miR-130a upregulated the expression of type I IFN (IFN-a/ IFN -b), ISG15, USP18 and MxA, which are involved in innate immune response and decreased expression of miR122, a well-defined miRNA promoting HCV production. In conclusion, miR-130a inhibits HCV replication/production by restoring host innate immune responses and/or downregulating pro-HCV miR-122. miR-130a might be a potential drug target by modulating host innate immune responses to combat HCV infection. Keywords: hepatitis C virus, innate immune response, interferon-stimulated genes, microRNA-122, microRNA130a.
with promising early results, there remains a subset of ‘difficult to treat’ patients who still require IFN-a-based strategies. For these reasons, there is an obvious need to better understand how the IFN-a-dependent innate immune response can be manipulated to optimize patient care. miRNAs are endogenous noncoding small RNAs of 18– 25 nucleotides that regulate gene expression at the posttranscription level [6–8]. By binding to complementary binding sites within the 3′ untranslated region (3′ UTR) of target mRNAs, miRNAs impair their translation or promote their degradation and in so doing control diverse cellular processes [9]. Recent studies have demonstrated that human miRNAs can affect viral replication. In HCV, the most noteworthy discovery to date is that miR-122 promotes HCV production [10]. However, other miRNAs are likely to contribute to the viral life cycle. Most recently, by infecting na€ıve Huh7.5.1 cells with a relatively high titre of JFH1 virus, Liu et al. used microarray techniques to identify 22 upregulated and 20 downregulated miRNAs in response to HCV infection [11,12]. In this study, miR130a was significantly downregulated after HCV infection [11]. Peng et al. [12] carried out a computational study of hepatitis C virus–associated miRNAs–mRNA regulatory modules in human livers and identified 38 miRNA–mRNA regulatory modules in the network that were associated
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with HCV infection. Contrary to the previous study, their results suggested that miR-130 was upregulated in the HCV-infected liver tissues. Still, another study implied that miR-130a might be a potential regulator associated with HCV infection [13]. In this research, authors found that the expression of miR-130a was upregulated about 2.2-fold [13]. Although the results are somewhat conflicting, nevertheless there is reason to think that miRNA plays a role in HCV infection and that the nature and mechanism of its role should be determined. In contrast to other virus, HCV is a poor inducer of IFN and pro-inflammatory cytokines in cell culture systems. One reason for this is that the HCV NS3/4A protease cleaves MAVS, an essential adaptor protein for RIG-Iinduced IFN signalling pathway, resulting in a rapid disruption of the function of MAVS and in abrogation of the IFN induction pathway [14]. Due to RIG-I mutation (a T55I substitution in the first CARD domain of RIG-I) resulting in abolished IFN induction following virus infection in Huh7.5 cells, HCV replicates very efficiently in these cells [15]. In the current study, we investigate the role of miR130a in HCV production in both HCV replicon and JFH1 HCVcc systems. Our data demonstrate that upregulating miR-130a by transfection with a miR-130a mimic significantly inhibits HCV replication in both HCV replicon and JFH1-infected cells. Additionally, type I IFN (IFN-a/IFN-b), ISG15, USP18 and MxA, all proteins that are involved in the host innate immune response, are greatly upregulated by increased miR-130a. Furthermore, we also found that overexpression of miR-130a decreased expression of miR122, a well-defined miRNA promoting HCV production. These results suggest that miR-130a inhibits HCV replication/production by restoring host innate immune responses and/or downregulating pro-HCV miR-122. miR-130a might be a potential drug target by modulating host innate immune responses to combat HCV infection.
MATERIALS AND METHODS Cell culture and virus The Huh-7.5.1 cell line was kindly provided by Professor Zhongtian Qi (the Second Military Medical University, Shanghai, China). Con1b subgenomic genotype 1b HCV replicon cell line was obtained from Dr. Ian McGilvray (University of Toronto, Canada). The Con1b cell line is a Huh7.5 cell population containing the full-length HCV genotype 1b replicon. The cells were maintained in DMEM supplemented with 10% (v/v) FBS, 100 units/mL penicillin and streptomycin, 100 lg/mL nonessential amino acid and selection antibiotic G418 (1000 lg/mL) for Con1b cells. HCV infectious clone J6/JFH1, the full-length chimerical genome from the infectious JFH1 (genotype 2a), was generously provided by Dr. Charles Rice (Rockfeller University).
MicroRNA miR-130a (miRIDIAN mimic hsa-mir-130a; C-300598-030005) and negative-control miRNA (miRIDIAN mimic negative control; CN-001000-01) were purchased from Dharmacon (Lafayette, CO, USA).
In vitro transfection and infection Cells were seeded in 24-well plates at 2 9 105 cells per well for 24 h. Transfection with miR-130a mimic or miRNA mimic negative control (MMNC) (final concentration 2 nM) was performed using DharmaFECT4 (Dharmacon) according to the manufacturer’s instruction. Cells were harvested 48 h post–transfection, and total RNA was extracted as described below. To infect Huh7.5.1 cells, 50 lL of HCV J6/JFH1 stock (1.6 9 105 TCID50/mL –MOI = 0.56) was added into each well for 4 h, after which the culture medium was removed and the cells washed twice with PBS before transfection.
IFN-a treatment experiment Cells were seeded in 24-well plates at 1 9 105 cells per well for 24 h and then 0, 10, 100, 300 or 1000 IU/mL IFN-a was added in each well in triplicate. Cells were harvested 48 h later, and total RNA was extracted as described below.
RNA extraction Total RNA was isolated using TRIzol method (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. In brief, culture media were removed and 1 mL TRIzol regent was added per 10 cm2 of culture dish surface area. After cell lysis, chloroform was added (0.2 mL/1 mL TRIzol), and the mixture centrifuged to separate it into three phases. The upper aqueous phase was isolated and placed in a fresh tube. 100% isopropanol was added (0.5 mL/1 mL TRIzol), and the mixture centrifuged to get an RNA-rich pellet. The RNA pellet was washed with 75% ethanol, and the total RNA was resuspended in 20 lL RNase-free H2O. RNA concentration was obtained by spectrometry at OD 260.
Reverse transcription and real-time PCR The first-strand complementary DNA (cDNA) was synthesized for gene expression analysis and miRNA expression using random primer (Roche, Basel, Switzerland) and miR-130a-specific reverse transcription primer (RiboBio Co, Ltd., Guangzhou, China), respectively. The real-time RT-PCR for the quantification of HCV, IFN-a, IFN-b, ISG15, USP18, MxA, SOCS3 and GAPDH mRNAs was performed with the FastStart Universal SYBR Green © 2013 John Wiley & Sons Ltd
miR-130a inhibits HCV replication in vitro Table 1 Real-time PCR primers
Gene
Sequence
HCV Con1b
Forward 5′-GAAAGCGTCTAGCCAT-3′ Reverse 5′-CTCGCAAGCACCCTATCAG-3′ Forward 5′-GCAGAAAGCGCCTAGCCAT-3′ Reverse 5′-CTCGCAAGCGCCCTATCAG-3′ Forward 5′-GCCTCCTGCACCACCAACTG-3′ Reverse 5′-ACGCCTGCTTCACCACCTTC-3′ Forward 5′-TCGCCCTTTGCTTTACTGAT-3′ Reverse 5′-GGGTCTCAGGGAGATCACAG-3′ Forward 5′-AAACTC ATAGCAGTCTGCA-3′ Reverse 5′-AGGAGATCTTCAGTTTCGGAGG-3′ Forward 5′-CGCAGATCACCCAGAAGATT-3′ Reverse 5′-GCCCTTGTTATTCCTCACCA-3′ Forward 5′-CAGACCCTGACAATCCACCT-3′ Reverse 5′-AGCTCATACTGCCCTCCAGA-3′ Forward 5′-GTGCATTGCAGAAGGTCAGA-3′ Reverse 5′-CTGGTGATAGGCCATCAGGT-3′
HCV JFH-1 GAPDH IFN-a IFN-b ISG15 USP18 MxA
Master Mix (Roche). GAPDH mRNA levels were used as an endogenous reference to normalize the quantities of target mRNA. The primers used in this study are listed in Table 1. Quantification of miR-130a and miR-122 was performed using miR-130a-specific and miR-122-specific primers normalized to U6 as an internal control.
Statistical analyses Data were statistically analysed by an ANOVA with post hoc analysis, and P values less than 0.05 were considered statistically significant. All data are representative of at least three repeated experiments.
RESULTS miR-130a overexpression suppresses HCV RNA replication in both the Con1b replicon and the HCV JFH1-based cell culture system To determine the role of miR-130a in HCV RNA replication, HCV replicon cells (Con1b) were transfected with 2 nM miR-130a mimic or miRNA mimic negative control (MMNC) and HCV RNAs were quantified by quantitative RT-PCR 48 h post-transfection. As shown in Fig. 1, miR-130a levels were significantly upregulated following transfection and 2 nM of miR-130a mimic resulted in a significant reduction in HCV RNA replication compared with the same concentration of miRNA mimic negative control (P < 0.05). We observed a similar inhibitory effect of miR-130a on HCV RNA replication in the HCV J6/JFH1 cell culture system. 2 nM miR-130a led to a significant decrease in HCV JFH1 RNA (P < 0.05 compared with negative control, Fig. 1d). © 2013 John Wiley & Sons Ltd
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Length 241 bp 244 bp 349 bp 82 bp 168 bp 185 bp 164 bp 140 bp
miR-130a overexpression increased endogenous IFN-a and IFN-b production The host innate immune response, mediated by production of endogenous interferon (IFN-a/IFN-b), is the first line of defence following virus infection. Having established that increased levels of miR-130a lead to decreased HCV replication, we next determined whether the antiHCV effect of miR-130a was associated with upregulation of endogenous IFN-a and IFN-b. As shown in Fig. 2, the expression of IFN-a and IFN-b was increased significantly both in Con1b replicon and in the HCV JFH1-based cell culture system after transfection of miR-130a mimic into cells. In Con1b replicon, overexpression of miR-130a increased IFN-a by 29-fold and IFN-b by 52-fold compared with miRNA mimic negative control. A similar trend was observed in HCVcc JFH1-based cell culture system although the effect was less (2.3-fold and 2.5-fold for IFN-a and IFN-b, respectively). Because Huh7.5.1 cells we used for this study are deficient in both TLR3 and RIG-I, two most important mediators inducing type I IFN production, normally these cells cannot recognize HCV infection and as a result, almost no type I IFN is produced following virus infection. Based on the results that overexpression of miR-130a stimulated type I IFN production in both replicon and J6-/JFH1-infected cells, we concluded that miR-130a restores the innate immune response to produce type I IFNs. To test whether miR-130a is interferon inducible, we stimulated Huh7 and Huh7.5.1 cells with various dosage of exogenous IFN-a. Low-dose IFN-a did not affect the expression levels of cellular miR-130a in both Huh7 and Huh7.5.1 cells. In Huh7.5.1 cells, the miR-130a was downregulated dramatically when cells were treated with
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Fig. 1 miR-130a inhibits HCV replication in both replicon (1b) and J6/JFH1 culture systems. (a) miR-130a overexpression after transfection of miR-130a mimic in HCV Con1b replicon cells. (b) miR-130a overexpression in JFH-1 HCVcc system. (c) Overexpression of miR-130a inhibited HCV RNA replication in Con1b replicon cells. (d) Overexpression of miR-130a inhibited HCV RNA replication in JFH-1 HCVcc system. Cells were transfected for 48 h with 2 nM miR-130a mimic or microRNA mimic negative control (MMNC) by DharmaFECT4 (Dharmacon), after which cells were harvested and total RNA was extracted. The levels of miR-130a or HCV RNA and host cell U6 or GAPDH mRNA were measured by quantitative RT-PCR as described in Materials and Methods. Data are presented as means SEM, n = 3. Error bars indicate standard error of mean (SEM). *P < 0.05; **P < 0.01 vs control and mock and MMNC (microRNA mimic negative control).
high concentration of IFN-a at 1000 IU/mL (Fig. 3), but this effect may be due to the toxic effect of high-dose IFN.
system, respectively. In line with this data, mRNA levels of two additional ISGs, ISG15 and USP18, were also increased in HCVcc JFH1-based cell culture system (Fig. 4).
miR-130a upregulated the downstream interferon-stimulated genes (ISGs) expression
miR-130a downregulated the expression of miR-122
Because the type I IFNs (IFN-a and IFN-b) activate JAK/ STAT signalling pathway and lead to upregulation of several hundred IFN-stimulated genes (ISGs) within the cell [16], we next determined whether upregulation of IFN-a/ IFN-b by miR-130a could increase ISGs expression in both Con1b- and JFH1-infected cells. If the inhibitory effects of increased miR-130a on HCV replication are mediated via increased IFN signalling, various ISGs at the downstream IFN signalling pathway should be increased. As expected, miR-130a significantly upregulated MxA mRNA by 4.65- and 2.1-fold (Fig. 4), compared with the same amount of miRNA mimic negative control, in the Con1b replicon and HCVcc JFH1-based cell culture
miR-122 is a well-known liver-specific microRNA that positively regulates hepatitis C virus (HCV) RNA abundance and is essential for production of infectious HCV [17]. So, we next studied whether the inhibition effect on HCV of miR-130a was related to miR-122. As shown in Fig. 5, overexpression of miR-130a decreased the miR-122 expression significantly both in Con1b replicon and in JFH1 HCVcc culture system. As it was reported that IFN-a/IFN-b treatment of the human hepatocyte cell line Huh7 leads to a temporary attenuation of miR-122 expression by 20– 40% [18], miR-130a may suppress HCV replication through restoring IFN-a production in Huh7.5.1 cells to downregulate miR-122 indirectly. © 2013 John Wiley & Sons Ltd
miR-130a inhibits HCV replication in vitro (a)
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Fig. 2 miR-130a restores innate immune system to produce type I IFNs. (a) Overexpression of miR-130a increased endogenous IFN-a expression in HCV Con1b replicon. (b) Overexpression of miR-130a increased endogenous IFN-b expression in HCV Con1b replicon. (c) Overexpression of miR-130a increased endogenous IFN-a expression in JFH-1 HCVcc. (d) Overexpression of miR-130a increased endogenous IFN-b expression in JFH-1 HCVcc. Cells were transfected for 48 h with 2 nM miR-130a mimic or microRNA mimic negative control (MMNC) by DharmaFECT4 (Dharmacon), after which cells were harvested and total RNA was extracted. The levels of IFN-a or IFN-b and host cell GAPDH mRNA were measured by quantitative RT-PCR as described in Materials and Methods. Data are presented as means SEM, n = 3. Error bars indicate standard error of mean (SEM). *P < 0.05; **P < 0.01 vs control and mock and MMNC (microRNA mimic negative control).
DISCUSSION There have been several reports documenting the inhibition of viral replication by endogenous cellular miRNA species. miR-32 was reported to inhibit the primate foamy virus (PFV) replication in the human embryonic kidney cell line 293T [19]. HIV-1 replication was significantly inhibited by cellular miR-29a in T cells [20]. However, the mechanisms by which the miRNAs modulate the viral replication are still unknown. While some miRNAs could directly targeting viral mRNA species, cellular miRNAs may also function by modulating the expression of cellular factors related to viral replication or host innate immune responses [18,19]. Using HCV replicon and HCVcc culture system, we demonstrated that HCV replication can be inhibited by miR-130a through restoring type I IFN production and/or suppressing pro-HCV miRNA-122 expression. The results presented here support a link between HCV replication and altered expression of miR-130a targeting © 2013 John Wiley & Sons Ltd
host innate immune responses. Endogenous type I interferons (IFNs) are the main antiviral cytokines [21]. IFN-a/ IFN-b is produced and binds to their receptors, signals through the Jak-STAT pathway and activates a few hundred target genes, collectively called interferon-stimulated genes (ISGs). Many antiviral ISGs degrade viral RNAs and block their translation [21]. However, HCV interferes with Jak/STAT pathway to evade IFN-induced innate immune response [22]. Our results showed that overexpression of miR-130a results in increased expression of IFN-a/IFN-b and the IFN-stimulated genes (ISGs), including MxA, ISG15 and USP18. Interestingly, expression levels of type I IFNs (Fig. 2) in Con1b replicon cells are higher than those in JFH1 HCVcc, but downstream ISG (Fig. 4) and miRNA122 (Fig. 5) expression levels are similar in two systems. In fact, Con1b cell (replicon) contains HCV RNA only but JFH1 HCVcc can produce full life cycle of HCV, which contains not only HCV RNA but also viral proteins. Some viral protein, such as NS3/4A protease, has been proven to be able to inhibit type I IFN (IFN-a, IFN-b)
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Fig. 3 IFN-a stimulus does not affect the expression of cellular miR-130a in Huh7 and Huh7.5.1 cells. Cells were stimulated with IFN-a (10–1000 IU/mL) for 24 h, and total RNA was extracted. The levels of cellular miR-130a and U6 were measured by quantitative RT-PCR as described in Materials and Methods. Data are presented as means SEM, n = 3. Error bars indicate standard error of mean (SEM).
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production, which may explain less induction of IFN-a and IFN-b in JFH1 HCVcc-infected cells. The downstream ISG induction is similar in two systems regardless of IFN levels, indicating the possibility of IFN resistance due to activation of the negative regulators of IFN signalling, such as SOCC1 and SOCS3. Huh7.5.1 cells have a point mutation at the threonine 55 residue of RIG-I and therefore unable to sense the HCV infection to produce IFNs. Our results provide evidence that upregulation of miR-130a restores the IFN production in Huh7.5.1 cells. As a result, IFN-induced ISGs inhibit the replication of HCV RNA. The restoration of innate immune response to produce type I IFNs in both HCV replicon and J6/JFH1 cells seems to be microRNA
Fig. 4 Overexpression of miR-130a stimulates ISGs expression. (a) Overexpression of miR-130a increased MxA expression in HCV Con1b replicon. (b) Overexpression of miR130a increased MxA expression in JFH1 HCVcc. (c) Overexpression of miR130a increased ISG15 expression in JFH1 HCVcc. (d) Overexpression of miR130a increased USP18 expression in JFH1 HCVcc. Cells were transfected for 48 h with 2 nM miR-130a mimic or microRNA mimic negative control (MMNC) by DharmaFECT4 (Dharmacon), after which cells were harvested and total RNA was extracted. The levels of MxA, ISG15, USP18 and host cell GAPDH mRNA were measured by quantitative RT-PCR as described in Materials and Methods. Data are presented as means SEM, n = 3. Error bars indicate standard error of mean (SEM). *P < 0.05;**P < 0.01 vs control and mock and MMNC (microRNA mimic negative control).
specific, because another microRNA (miR-146a) has no such effect (data not shown). Because high-dose type I IFNs can overcome the HCV interference with Jak/STAT signalling pathway, miR-130a-induced upregulation of IFN-a/IFN-b may contribute to the virus spontaneous clearance, making it a potential strategy for new antiviral development. Recently, using microarray technology, Pedersen et al. [18] identified eight miRNAs (miR-1, miR-30, miR-128, miR-196, miR-296, miR-351, miR-431 and miR-448) that are able to inhibit HCV replication and infection and all these miRNAs are type I IFN inducible. Similarly, it was reported that IFN-b stimulates miR-155 expression [23]. © 2013 John Wiley & Sons Ltd
miR-130a inhibits HCV replication in vitro (a)
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Fig. 5 Overexpression of miR-130a decreased miR-122 expression in HCV Con1b replicon and JFH-1 HCVcc. Cells were transfected for 48 h with 2 nM miR-130a mimic or microRNA mimic negative control (MMNC) by DharmaFECT4 (Dharmacon), after which cells were harvested and total RNA was extracted. The levels of miR-122 and host cell U6 were measured by quantitative RT-PCR as described in Materials and Methods. Data are presented as means SEM, n = 3. Error bars indicate standard error of mean (SEM). **P < 0.01 vs control and mock and MMNC (microRNA mimic negative control). These results provide evidence that modulating cellular miRNAs may be one of the mechanisms that interferon system combat viral infection. Interestingly, we found that miR-130a is not IFN inducible yet can upregulate the expression of IFN-a/IFN-b. Our results extend previous investigations into the modulation of the IFN system and, specifically, the ability of miRNA to regulate type I IFN expression. miR-122, which is liver specific, was confirmed to bind directly to both sides at the 5′ untranslated region (5′ UTR) of HCV RNA and positively regulates the viral life cycle [24]. Using a high-affinity locked nucleic acid-modified (LNA-modified) anti-miR-122, Elmen et al. silenced miR-122 efficiently in African green monkeys, leading to a dose-dependent and long-lasting decrease in serum cholesterol [25,26]. Recently, they injected the anti-miR-122 into chimpanzees chronic infected by genotype 1 HCV and acquired long-lasting suppression of HCV viraemia with no viral resistance or side effects [26,27]. Our results showed that the expression of miR-122 was downregulated after transfecting miR-130a in both Con1b replicon cell line and JFH-1 HCVcc system. As inhibition of miR-122 presents a very attractive novel approach to treat HCV, miR-130a may be a good potential strategy. It was reported that IFNa can suppress miR-122 expression [18] and upregulation of miR-130a restores IFN-a production, downregulation of
miR-122 expression by miR-130a may be mediated by increased IFN-a production. Overall, the study suggests that miR-130a inhibits HCV RNA replication in human hepatocytes by restoring IFN production and increasing the expression of the downstream anti-HCV ISGs. Meanwhile, miR-130a acts as a native antagonist of miR-122, presumably through increased expression of IFN. All of these contribute to the inhibitory effect of miR-130a on HCV replication, representing a novel strategy for HCV therapy.
ACKNOWLEDGEMENTS We thank Dr. Zhongtian Qi for providing Huh7.5.1 cells and Dr. Charles Rice for the J6/JFH1 HCVcc system.
FINANCIAL SUPPORT This study was supported by grants from the Sichuan Provincial Science and Technology department (2011SZ0010 to Dr. L Chen and 2013JY0048 to Dr. S Li).
CONFLICT OF INTEREST No conflict of interests exists for all authors.
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© 2013 John Wiley & Sons Ltd
doi:10.1111/jvh.12131
MicroRNA-130a inhibits HCV replication by restoring the innate immune response S. Li,1,* X. Duan,1,* Y. Li,1 B. Liu,1 I. McGilvray1,2 and L. Chen1,2
1
The Institute of Blood Transfusion,
2
Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China; and Toronto General Research Institute, University of Toronto, Toronto, ON, Canada Received March 2013; accepted for publication May 2013
SUMMARY. Hepatitis C virus (HCV) infection is a major
cause of chronic hepatitis and hepatocellular carcinoma. Currently pegylated interferon (IFN) combined with ribavirin remains the best therapeutic approach, although patients infected with HCV genotype I may benefit from adding protease inhibitors as ‘triple therapy’. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression and have recently been shown to play an important role in human innate immune response and as an antiviral in chimpanzees. We studied the effect of miR-130a on the HCV replication. We found that miR130a significantly inhibits HCV replication in both HCV replicon and J6-/JFH1-infected cells. Over expression of
INTRODUCTION Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) [1]. There are about 170 million people infected with HCV worldwide. Although direct-acting antivirals (DAAs) targeting HCV polymerases or proteases are now available, interferon alpha (IFN-a) combined with ribavirin remains the standard care for treatment for chronic HCV infection in most developing countries [2].There is good reason to think that IFN-a will remain part of the treatment regimen for many years to come. HCV is an extremely mutagenic virus, and resistance has been described to every DAA to date [3–5]. Although IFN-a-free combinations of DAAs have been attempted Abbreviations: DAA, direct-acting antivirals; HCC, hepatocellular carcinoma; ISGs, interferon-stimulated genes; LNA, locked nucleic acid; MMNC, miRNA mimic negative control; PFV, primate foamy virus; UTR, untranslated region. Correspondence: Dr. Limin Chen, The Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, Sichuan, China. E-mail: [email protected] *These authors contributed equally to this study.
© 2013 John Wiley & Sons Ltd
miR-130a upregulated the expression of type I IFN (IFN-a/ IFN -b), ISG15, USP18 and MxA, which are involved in innate immune response and decreased expression of miR122, a well-defined miRNA promoting HCV production. In conclusion, miR-130a inhibits HCV replication/production by restoring host innate immune responses and/or downregulating pro-HCV miR-122. miR-130a might be a potential drug target by modulating host innate immune responses to combat HCV infection. Keywords: hepatitis C virus, innate immune response, interferon-stimulated genes, microRNA-122, microRNA130a.
with promising early results, there remains a subset of ‘difficult to treat’ patients who still require IFN-a-based strategies. For these reasons, there is an obvious need to better understand how the IFN-a-dependent innate immune response can be manipulated to optimize patient care. miRNAs are endogenous noncoding small RNAs of 18– 25 nucleotides that regulate gene expression at the posttranscription level [6–8]. By binding to complementary binding sites within the 3′ untranslated region (3′ UTR) of target mRNAs, miRNAs impair their translation or promote their degradation and in so doing control diverse cellular processes [9]. Recent studies have demonstrated that human miRNAs can affect viral replication. In HCV, the most noteworthy discovery to date is that miR-122 promotes HCV production [10]. However, other miRNAs are likely to contribute to the viral life cycle. Most recently, by infecting na€ıve Huh7.5.1 cells with a relatively high titre of JFH1 virus, Liu et al. used microarray techniques to identify 22 upregulated and 20 downregulated miRNAs in response to HCV infection [11,12]. In this study, miR130a was significantly downregulated after HCV infection [11]. Peng et al. [12] carried out a computational study of hepatitis C virus–associated miRNAs–mRNA regulatory modules in human livers and identified 38 miRNA–mRNA regulatory modules in the network that were associated
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with HCV infection. Contrary to the previous study, their results suggested that miR-130 was upregulated in the HCV-infected liver tissues. Still, another study implied that miR-130a might be a potential regulator associated with HCV infection [13]. In this research, authors found that the expression of miR-130a was upregulated about 2.2-fold [13]. Although the results are somewhat conflicting, nevertheless there is reason to think that miRNA plays a role in HCV infection and that the nature and mechanism of its role should be determined. In contrast to other virus, HCV is a poor inducer of IFN and pro-inflammatory cytokines in cell culture systems. One reason for this is that the HCV NS3/4A protease cleaves MAVS, an essential adaptor protein for RIG-Iinduced IFN signalling pathway, resulting in a rapid disruption of the function of MAVS and in abrogation of the IFN induction pathway [14]. Due to RIG-I mutation (a T55I substitution in the first CARD domain of RIG-I) resulting in abolished IFN induction following virus infection in Huh7.5 cells, HCV replicates very efficiently in these cells [15]. In the current study, we investigate the role of miR130a in HCV production in both HCV replicon and JFH1 HCVcc systems. Our data demonstrate that upregulating miR-130a by transfection with a miR-130a mimic significantly inhibits HCV replication in both HCV replicon and JFH1-infected cells. Additionally, type I IFN (IFN-a/IFN-b), ISG15, USP18 and MxA, all proteins that are involved in the host innate immune response, are greatly upregulated by increased miR-130a. Furthermore, we also found that overexpression of miR-130a decreased expression of miR122, a well-defined miRNA promoting HCV production. These results suggest that miR-130a inhibits HCV replication/production by restoring host innate immune responses and/or downregulating pro-HCV miR-122. miR-130a might be a potential drug target by modulating host innate immune responses to combat HCV infection.
MATERIALS AND METHODS Cell culture and virus The Huh-7.5.1 cell line was kindly provided by Professor Zhongtian Qi (the Second Military Medical University, Shanghai, China). Con1b subgenomic genotype 1b HCV replicon cell line was obtained from Dr. Ian McGilvray (University of Toronto, Canada). The Con1b cell line is a Huh7.5 cell population containing the full-length HCV genotype 1b replicon. The cells were maintained in DMEM supplemented with 10% (v/v) FBS, 100 units/mL penicillin and streptomycin, 100 lg/mL nonessential amino acid and selection antibiotic G418 (1000 lg/mL) for Con1b cells. HCV infectious clone J6/JFH1, the full-length chimerical genome from the infectious JFH1 (genotype 2a), was generously provided by Dr. Charles Rice (Rockfeller University).
MicroRNA miR-130a (miRIDIAN mimic hsa-mir-130a; C-300598-030005) and negative-control miRNA (miRIDIAN mimic negative control; CN-001000-01) were purchased from Dharmacon (Lafayette, CO, USA).
In vitro transfection and infection Cells were seeded in 24-well plates at 2 9 105 cells per well for 24 h. Transfection with miR-130a mimic or miRNA mimic negative control (MMNC) (final concentration 2 nM) was performed using DharmaFECT4 (Dharmacon) according to the manufacturer’s instruction. Cells were harvested 48 h post–transfection, and total RNA was extracted as described below. To infect Huh7.5.1 cells, 50 lL of HCV J6/JFH1 stock (1.6 9 105 TCID50/mL –MOI = 0.56) was added into each well for 4 h, after which the culture medium was removed and the cells washed twice with PBS before transfection.
IFN-a treatment experiment Cells were seeded in 24-well plates at 1 9 105 cells per well for 24 h and then 0, 10, 100, 300 or 1000 IU/mL IFN-a was added in each well in triplicate. Cells were harvested 48 h later, and total RNA was extracted as described below.
RNA extraction Total RNA was isolated using TRIzol method (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. In brief, culture media were removed and 1 mL TRIzol regent was added per 10 cm2 of culture dish surface area. After cell lysis, chloroform was added (0.2 mL/1 mL TRIzol), and the mixture centrifuged to separate it into three phases. The upper aqueous phase was isolated and placed in a fresh tube. 100% isopropanol was added (0.5 mL/1 mL TRIzol), and the mixture centrifuged to get an RNA-rich pellet. The RNA pellet was washed with 75% ethanol, and the total RNA was resuspended in 20 lL RNase-free H2O. RNA concentration was obtained by spectrometry at OD 260.
Reverse transcription and real-time PCR The first-strand complementary DNA (cDNA) was synthesized for gene expression analysis and miRNA expression using random primer (Roche, Basel, Switzerland) and miR-130a-specific reverse transcription primer (RiboBio Co, Ltd., Guangzhou, China), respectively. The real-time RT-PCR for the quantification of HCV, IFN-a, IFN-b, ISG15, USP18, MxA, SOCS3 and GAPDH mRNAs was performed with the FastStart Universal SYBR Green © 2013 John Wiley & Sons Ltd
miR-130a inhibits HCV replication in vitro Table 1 Real-time PCR primers
Gene
Sequence
HCV Con1b
Forward 5′-GAAAGCGTCTAGCCAT-3′ Reverse 5′-CTCGCAAGCACCCTATCAG-3′ Forward 5′-GCAGAAAGCGCCTAGCCAT-3′ Reverse 5′-CTCGCAAGCGCCCTATCAG-3′ Forward 5′-GCCTCCTGCACCACCAACTG-3′ Reverse 5′-ACGCCTGCTTCACCACCTTC-3′ Forward 5′-TCGCCCTTTGCTTTACTGAT-3′ Reverse 5′-GGGTCTCAGGGAGATCACAG-3′ Forward 5′-AAACTC ATAGCAGTCTGCA-3′ Reverse 5′-AGGAGATCTTCAGTTTCGGAGG-3′ Forward 5′-CGCAGATCACCCAGAAGATT-3′ Reverse 5′-GCCCTTGTTATTCCTCACCA-3′ Forward 5′-CAGACCCTGACAATCCACCT-3′ Reverse 5′-AGCTCATACTGCCCTCCAGA-3′ Forward 5′-GTGCATTGCAGAAGGTCAGA-3′ Reverse 5′-CTGGTGATAGGCCATCAGGT-3′
HCV JFH-1 GAPDH IFN-a IFN-b ISG15 USP18 MxA
Master Mix (Roche). GAPDH mRNA levels were used as an endogenous reference to normalize the quantities of target mRNA. The primers used in this study are listed in Table 1. Quantification of miR-130a and miR-122 was performed using miR-130a-specific and miR-122-specific primers normalized to U6 as an internal control.
Statistical analyses Data were statistically analysed by an ANOVA with post hoc analysis, and P values less than 0.05 were considered statistically significant. All data are representative of at least three repeated experiments.
RESULTS miR-130a overexpression suppresses HCV RNA replication in both the Con1b replicon and the HCV JFH1-based cell culture system To determine the role of miR-130a in HCV RNA replication, HCV replicon cells (Con1b) were transfected with 2 nM miR-130a mimic or miRNA mimic negative control (MMNC) and HCV RNAs were quantified by quantitative RT-PCR 48 h post-transfection. As shown in Fig. 1, miR-130a levels were significantly upregulated following transfection and 2 nM of miR-130a mimic resulted in a significant reduction in HCV RNA replication compared with the same concentration of miRNA mimic negative control (P < 0.05). We observed a similar inhibitory effect of miR-130a on HCV RNA replication in the HCV J6/JFH1 cell culture system. 2 nM miR-130a led to a significant decrease in HCV JFH1 RNA (P < 0.05 compared with negative control, Fig. 1d). © 2013 John Wiley & Sons Ltd
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Length 241 bp 244 bp 349 bp 82 bp 168 bp 185 bp 164 bp 140 bp
miR-130a overexpression increased endogenous IFN-a and IFN-b production The host innate immune response, mediated by production of endogenous interferon (IFN-a/IFN-b), is the first line of defence following virus infection. Having established that increased levels of miR-130a lead to decreased HCV replication, we next determined whether the antiHCV effect of miR-130a was associated with upregulation of endogenous IFN-a and IFN-b. As shown in Fig. 2, the expression of IFN-a and IFN-b was increased significantly both in Con1b replicon and in the HCV JFH1-based cell culture system after transfection of miR-130a mimic into cells. In Con1b replicon, overexpression of miR-130a increased IFN-a by 29-fold and IFN-b by 52-fold compared with miRNA mimic negative control. A similar trend was observed in HCVcc JFH1-based cell culture system although the effect was less (2.3-fold and 2.5-fold for IFN-a and IFN-b, respectively). Because Huh7.5.1 cells we used for this study are deficient in both TLR3 and RIG-I, two most important mediators inducing type I IFN production, normally these cells cannot recognize HCV infection and as a result, almost no type I IFN is produced following virus infection. Based on the results that overexpression of miR-130a stimulated type I IFN production in both replicon and J6-/JFH1-infected cells, we concluded that miR-130a restores the innate immune response to produce type I IFNs. To test whether miR-130a is interferon inducible, we stimulated Huh7 and Huh7.5.1 cells with various dosage of exogenous IFN-a. Low-dose IFN-a did not affect the expression levels of cellular miR-130a in both Huh7 and Huh7.5.1 cells. In Huh7.5.1 cells, the miR-130a was downregulated dramatically when cells were treated with
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Fig. 1 miR-130a inhibits HCV replication in both replicon (1b) and J6/JFH1 culture systems. (a) miR-130a overexpression after transfection of miR-130a mimic in HCV Con1b replicon cells. (b) miR-130a overexpression in JFH-1 HCVcc system. (c) Overexpression of miR-130a inhibited HCV RNA replication in Con1b replicon cells. (d) Overexpression of miR-130a inhibited HCV RNA replication in JFH-1 HCVcc system. Cells were transfected for 48 h with 2 nM miR-130a mimic or microRNA mimic negative control (MMNC) by DharmaFECT4 (Dharmacon), after which cells were harvested and total RNA was extracted. The levels of miR-130a or HCV RNA and host cell U6 or GAPDH mRNA were measured by quantitative RT-PCR as described in Materials and Methods. Data are presented as means SEM, n = 3. Error bars indicate standard error of mean (SEM). *P < 0.05; **P < 0.01 vs control and mock and MMNC (microRNA mimic negative control).
high concentration of IFN-a at 1000 IU/mL (Fig. 3), but this effect may be due to the toxic effect of high-dose IFN.
system, respectively. In line with this data, mRNA levels of two additional ISGs, ISG15 and USP18, were also increased in HCVcc JFH1-based cell culture system (Fig. 4).
miR-130a upregulated the downstream interferon-stimulated genes (ISGs) expression
miR-130a downregulated the expression of miR-122
Because the type I IFNs (IFN-a and IFN-b) activate JAK/ STAT signalling pathway and lead to upregulation of several hundred IFN-stimulated genes (ISGs) within the cell [16], we next determined whether upregulation of IFN-a/ IFN-b by miR-130a could increase ISGs expression in both Con1b- and JFH1-infected cells. If the inhibitory effects of increased miR-130a on HCV replication are mediated via increased IFN signalling, various ISGs at the downstream IFN signalling pathway should be increased. As expected, miR-130a significantly upregulated MxA mRNA by 4.65- and 2.1-fold (Fig. 4), compared with the same amount of miRNA mimic negative control, in the Con1b replicon and HCVcc JFH1-based cell culture
miR-122 is a well-known liver-specific microRNA that positively regulates hepatitis C virus (HCV) RNA abundance and is essential for production of infectious HCV [17]. So, we next studied whether the inhibition effect on HCV of miR-130a was related to miR-122. As shown in Fig. 5, overexpression of miR-130a decreased the miR-122 expression significantly both in Con1b replicon and in JFH1 HCVcc culture system. As it was reported that IFN-a/IFN-b treatment of the human hepatocyte cell line Huh7 leads to a temporary attenuation of miR-122 expression by 20– 40% [18], miR-130a may suppress HCV replication through restoring IFN-a production in Huh7.5.1 cells to downregulate miR-122 indirectly. © 2013 John Wiley & Sons Ltd
miR-130a inhibits HCV replication in vitro (a)
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Fig. 2 miR-130a restores innate immune system to produce type I IFNs. (a) Overexpression of miR-130a increased endogenous IFN-a expression in HCV Con1b replicon. (b) Overexpression of miR-130a increased endogenous IFN-b expression in HCV Con1b replicon. (c) Overexpression of miR-130a increased endogenous IFN-a expression in JFH-1 HCVcc. (d) Overexpression of miR-130a increased endogenous IFN-b expression in JFH-1 HCVcc. Cells were transfected for 48 h with 2 nM miR-130a mimic or microRNA mimic negative control (MMNC) by DharmaFECT4 (Dharmacon), after which cells were harvested and total RNA was extracted. The levels of IFN-a or IFN-b and host cell GAPDH mRNA were measured by quantitative RT-PCR as described in Materials and Methods. Data are presented as means SEM, n = 3. Error bars indicate standard error of mean (SEM). *P < 0.05; **P < 0.01 vs control and mock and MMNC (microRNA mimic negative control).
DISCUSSION There have been several reports documenting the inhibition of viral replication by endogenous cellular miRNA species. miR-32 was reported to inhibit the primate foamy virus (PFV) replication in the human embryonic kidney cell line 293T [19]. HIV-1 replication was significantly inhibited by cellular miR-29a in T cells [20]. However, the mechanisms by which the miRNAs modulate the viral replication are still unknown. While some miRNAs could directly targeting viral mRNA species, cellular miRNAs may also function by modulating the expression of cellular factors related to viral replication or host innate immune responses [18,19]. Using HCV replicon and HCVcc culture system, we demonstrated that HCV replication can be inhibited by miR-130a through restoring type I IFN production and/or suppressing pro-HCV miRNA-122 expression. The results presented here support a link between HCV replication and altered expression of miR-130a targeting © 2013 John Wiley & Sons Ltd
host innate immune responses. Endogenous type I interferons (IFNs) are the main antiviral cytokines [21]. IFN-a/ IFN-b is produced and binds to their receptors, signals through the Jak-STAT pathway and activates a few hundred target genes, collectively called interferon-stimulated genes (ISGs). Many antiviral ISGs degrade viral RNAs and block their translation [21]. However, HCV interferes with Jak/STAT pathway to evade IFN-induced innate immune response [22]. Our results showed that overexpression of miR-130a results in increased expression of IFN-a/IFN-b and the IFN-stimulated genes (ISGs), including MxA, ISG15 and USP18. Interestingly, expression levels of type I IFNs (Fig. 2) in Con1b replicon cells are higher than those in JFH1 HCVcc, but downstream ISG (Fig. 4) and miRNA122 (Fig. 5) expression levels are similar in two systems. In fact, Con1b cell (replicon) contains HCV RNA only but JFH1 HCVcc can produce full life cycle of HCV, which contains not only HCV RNA but also viral proteins. Some viral protein, such as NS3/4A protease, has been proven to be able to inhibit type I IFN (IFN-a, IFN-b)
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Fig. 3 IFN-a stimulus does not affect the expression of cellular miR-130a in Huh7 and Huh7.5.1 cells. Cells were stimulated with IFN-a (10–1000 IU/mL) for 24 h, and total RNA was extracted. The levels of cellular miR-130a and U6 were measured by quantitative RT-PCR as described in Materials and Methods. Data are presented as means SEM, n = 3. Error bars indicate standard error of mean (SEM).
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production, which may explain less induction of IFN-a and IFN-b in JFH1 HCVcc-infected cells. The downstream ISG induction is similar in two systems regardless of IFN levels, indicating the possibility of IFN resistance due to activation of the negative regulators of IFN signalling, such as SOCC1 and SOCS3. Huh7.5.1 cells have a point mutation at the threonine 55 residue of RIG-I and therefore unable to sense the HCV infection to produce IFNs. Our results provide evidence that upregulation of miR-130a restores the IFN production in Huh7.5.1 cells. As a result, IFN-induced ISGs inhibit the replication of HCV RNA. The restoration of innate immune response to produce type I IFNs in both HCV replicon and J6/JFH1 cells seems to be microRNA
Fig. 4 Overexpression of miR-130a stimulates ISGs expression. (a) Overexpression of miR-130a increased MxA expression in HCV Con1b replicon. (b) Overexpression of miR130a increased MxA expression in JFH1 HCVcc. (c) Overexpression of miR130a increased ISG15 expression in JFH1 HCVcc. (d) Overexpression of miR130a increased USP18 expression in JFH1 HCVcc. Cells were transfected for 48 h with 2 nM miR-130a mimic or microRNA mimic negative control (MMNC) by DharmaFECT4 (Dharmacon), after which cells were harvested and total RNA was extracted. The levels of MxA, ISG15, USP18 and host cell GAPDH mRNA were measured by quantitative RT-PCR as described in Materials and Methods. Data are presented as means SEM, n = 3. Error bars indicate standard error of mean (SEM). *P < 0.05;**P < 0.01 vs control and mock and MMNC (microRNA mimic negative control).
specific, because another microRNA (miR-146a) has no such effect (data not shown). Because high-dose type I IFNs can overcome the HCV interference with Jak/STAT signalling pathway, miR-130a-induced upregulation of IFN-a/IFN-b may contribute to the virus spontaneous clearance, making it a potential strategy for new antiviral development. Recently, using microarray technology, Pedersen et al. [18] identified eight miRNAs (miR-1, miR-30, miR-128, miR-196, miR-296, miR-351, miR-431 and miR-448) that are able to inhibit HCV replication and infection and all these miRNAs are type I IFN inducible. Similarly, it was reported that IFN-b stimulates miR-155 expression [23]. © 2013 John Wiley & Sons Ltd
miR-130a inhibits HCV replication in vitro (a)
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Fig. 5 Overexpression of miR-130a decreased miR-122 expression in HCV Con1b replicon and JFH-1 HCVcc. Cells were transfected for 48 h with 2 nM miR-130a mimic or microRNA mimic negative control (MMNC) by DharmaFECT4 (Dharmacon), after which cells were harvested and total RNA was extracted. The levels of miR-122 and host cell U6 were measured by quantitative RT-PCR as described in Materials and Methods. Data are presented as means SEM, n = 3. Error bars indicate standard error of mean (SEM). **P < 0.01 vs control and mock and MMNC (microRNA mimic negative control). These results provide evidence that modulating cellular miRNAs may be one of the mechanisms that interferon system combat viral infection. Interestingly, we found that miR-130a is not IFN inducible yet can upregulate the expression of IFN-a/IFN-b. Our results extend previous investigations into the modulation of the IFN system and, specifically, the ability of miRNA to regulate type I IFN expression. miR-122, which is liver specific, was confirmed to bind directly to both sides at the 5′ untranslated region (5′ UTR) of HCV RNA and positively regulates the viral life cycle [24]. Using a high-affinity locked nucleic acid-modified (LNA-modified) anti-miR-122, Elmen et al. silenced miR-122 efficiently in African green monkeys, leading to a dose-dependent and long-lasting decrease in serum cholesterol [25,26]. Recently, they injected the anti-miR-122 into chimpanzees chronic infected by genotype 1 HCV and acquired long-lasting suppression of HCV viraemia with no viral resistance or side effects [26,27]. Our results showed that the expression of miR-122 was downregulated after transfecting miR-130a in both Con1b replicon cell line and JFH-1 HCVcc system. As inhibition of miR-122 presents a very attractive novel approach to treat HCV, miR-130a may be a good potential strategy. It was reported that IFNa can suppress miR-122 expression [18] and upregulation of miR-130a restores IFN-a production, downregulation of
miR-122 expression by miR-130a may be mediated by increased IFN-a production. Overall, the study suggests that miR-130a inhibits HCV RNA replication in human hepatocytes by restoring IFN production and increasing the expression of the downstream anti-HCV ISGs. Meanwhile, miR-130a acts as a native antagonist of miR-122, presumably through increased expression of IFN. All of these contribute to the inhibitory effect of miR-130a on HCV replication, representing a novel strategy for HCV therapy.
ACKNOWLEDGEMENTS We thank Dr. Zhongtian Qi for providing Huh7.5.1 cells and Dr. Charles Rice for the J6/JFH1 HCVcc system.
FINANCIAL SUPPORT This study was supported by grants from the Sichuan Provincial Science and Technology department (2011SZ0010 to Dr. L Chen and 2013JY0048 to Dr. S Li).
CONFLICT OF INTEREST No conflict of interests exists for all authors.
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