Molecular Mechanisms Of MiR146A Involved In HBV Infection Induced Inhibitory Response To Type â…  IFN In Hepatocytes

Object:HBV causes acute and chronic hepatitis and results in life-long infected, presenting a significant threat to public health. Interferon-α (IFN-α) has been used in the treatment of chronic HBV-related diseases for many years, but only30%of the patients show positive response to IFN-α, and many patients become resistant to IFN-α. This phenomenon is called "IFN-α resistance", which impairs IFN-α anti-HBV effect severely and therefore contributes to the damage, defect and disorder of host anti-viral immunity during HBV infectionIt is believed that HBV can impair anti-viral innate and adaptive immune response at molecular, cellular, organic and holistic levels, which lead to chronic infection to a certain extent. In the view of Liver immunology, activation of host anti-viral immunity is essential for HBV inhibition and clearance, and at the same time, is also required for desirable treatment effectiveness. Until now, research on negative immune regulation of HBV including "IFN resistance" is still delayed, which restricts the progress to develop new anti-HBV drugs.microRNAs (miRNAs), a big family of small non-coding RNAs, were found to serve the pivotal function of regulating gene expression at posttranscriptional level in recent years, which always inhibit mRNA translation by sequence-dependent "mis-match" in mammalian cells. It is reported that, both virus-and host-encoded miRNAs would be important regulators or executors involved in viral pathogenesis and immunity. During the whole period of HBV infection, miRNAs not only regulate replication, transcription and package itself, but also participate in liver inflammation, immune injury, latency, secondary cases such as hepatocellular carcinoma, which become a new viewpoint to explore human-HBV interaction.This research is designed to explore the role of miRNA in HBV-induced inhibition of innate immunity, especially "IFN resistance" by screening the expression level of immune-related miRNA between HBV-carried hepatoma cell and negative ones. We used many miRNA interference tools to analyze the interaction between HBV infection and miRNA changes, to determine whether miRNA was involved in the inhibition of intracellular innate immune pathway including "IFN resistance" after HBV infection. Using bioinformatic and molecular tools to look for key factors, we then identify the correlation between HBV and immune moleculars. At last, miRNA-targeting technology was employed to determine whether miRNA changes can favor anti-HBV response in HBV-carried mouse model.Methods:15immune-related miRNAs, including miR21, miR26a/b, miR106, miR132, miR146a/b, miR147, miR155, miR181, miR196a, miR296and miR448, were examined by stem-loop special miRNA qRT-PCR assay in HBV-carried HepG2.2.15cells and their counterpart HBV-negative HepG2cells. The miR146a expression levels were further confirmed by ribonuclease protection assay, also miR146a primary transcript (nearly200bp) and its stem-loop precursor (-100bp) were examined using special primers. Luciferase assay was used to test miRNA promoter activity.LMP-HBV1.2(a new HBV genome expression vector based on pAAV-HBV1.2) and polymerase (HBp), HBV core protein (HBc), HBx and Small HBV surface protein expression vectors based on pEGFP-N1were transfected into HepG2cells. HBV-carried BALB/c mice were established by hydrodynamic tail vein injection with pAAV-HBV1.2. After2weeks, serum was harvested, and HBsAg levels were detected by ELISA. Then primary mouse hepatocytes were isolated by collagenase perfusion. The effect of miR146a on HBV infection was determined by transferring HBV plasmid, and miR146a mimics, inhibitors or expression/silencing vectors in human hepatoma cells (HepG2, and HepG2.2.15) and in mouse by hydrodynamic tail vein injection. The change of miR146a was confirmed by stem-loop special miRNA qRT-PCR assay.In hepatoma cells, the effect of miR146a on IFN-a response was measured by RT-qPCR and Western Blotting, to test the transcription and translation including MxA、ISG15、OAS-1、IFIT3respectively. To explorer the mechanism of miR146a on STAT1expression, luciferase reporter vector containing STAT13’-UTR and its seed specific mutant control were constructed and cotransfected with miR146a mimics or inhibitors into hepatoma cells. The direct targeting effect of miR146a was validated by Luciferase reporter assay. STAT1protein level was also compared among HBV-positive and negative clinical samples.Bioinformatic analysis was carried out to look for new miR146a direct targets within17anti-viral factors, and3’-UTR of candidate targets were cloned to luciferase reporter vector for experiment validation. Luciferase assay was used to identify the exact target, IFIT3. Then, IFIT3and TRAF63’-UTR luciferase constructs and its seed specific mutant control were cotransfected into hepatoma cells to evaluate translation efficiency. The impact of miR146a on the translation of these two genes at posttranscriptional level was also estimated by luciferase assay. To determine the promotion of miR146a inhibition to type I interferon induction,5’ppp-RNA was transcripted in vitro, and then introduced into IIepG2.2.15cell after miR146a silencing by miR146a sponge vector. RT-qPCR of type I interferon was utilized to quantitate the increasing fold.Results:1. miR146a level was closely correlated to HBV infection in human hepatoma cellsTwo members of miR146family, miR146a and miR146b, showed a significant up-regulation in HepG2.2.15cells among15chosen miRNAs, especially miR146a. The expression of miR146a primary transcript and its stem-loop precursor were also higher in MepG2.2.15cells than that in HepG2cells. Promoter activity assay suggested that the transcription of miR146a was enhanced due to higher NF-κB activity.2. HBV infection induced miR146a expression both in vitro and in vivoStem-loop special miRNA qRT-PCR assay showed that miR146a was increased both in HepG2cells transfected with pAAV-HBV1.2transiently and in cells stabilizated by HBV genome vector. Four HBV main proteins all seemed to contribute to up-regulaion of miR146a when they were transfected into HepG2cells individually. The serum HBsAg level revealed a positive relationship with mature miR146a level in hepatocytes from HBV-carried mice.3. miR146a accelerated HBV replicationChemical synthesized miR146a mimics and inhibitors could interference endogenous level of miR146a effectively, as well as miR146a expression or silencing vector, pcDNA3-miR146a and psiRNE-PGK-miR146a-sponge.miR146a over-expression would ameliorate HBx and HBs/p mRNA transcription in HepG2cells. On the other hand, miR146a inhibitors down-regulated HBx and HBs/p mRNA levels in HepG2.2.15cells, as well as the secretion of HBeAg level in HepG2.2.15cell supernatant. And miR146a mimics showed the opposite influence on HBeAg secretion in HepG2.2.15cells. In vivo, mice co-injected with pAAV-HBV1.2and pcDNA3-miR146a plasmids by hydrodynamic tail vein injection showed higher level of serum HBsAg and HBeAg than that in pAAV-HBV1.2+pcDNA3treated mice.Bioinformatic analysis suggested that miR146a could hardly bind to HBV RNA directly.4. miR146a inhibited activation of type I interferon signalingmiR146a overexpression by miR146a mimics or expression vector decreased the sensitivity of HepG2cells to IFN-α stimulation, showing lower mRNA and protein level of several ISGs, including MxA, ISG15, OAS-1and IFIT3. On contrary, After HepG2.2.15cells were treated with miR146a inhibitor for24hours, the mRNA and protein levels of these genes were significantly increased in response to IFN-a.5. miR146a induced by HBV infection suppressed STAT1expression in hepatoma cellsThe basic level of STAT1was lower in HepG2.2.15cells than that in HepG2 cells and luciferase assay demonstrated STAT1was regulated at a post transcriptional level. The protein level of STAT1was up-regulated alter miR146a silenced in HepG2.2.15cells by treating with miR146a inhibitors, whereas down-regulated in HepG2cells treated with miR146a mimics with unchanged mRNA level. Reporter gene assay showed that miR146a exerted down-regulatory effect on STAT1via a sequence-dependent manner related to3"-UTR of STAT1mRNA in hepatoma cells. Additionally, STAT1protein level in HBV-positive samples of HCC patients was significantly lower than that in HBV-negative sample, implying the possibility of miR146a in suppressing STAT1expression.6. Human IFIT3is a potential target of miR146aIFIT3s R1G-I and RNase L were predicted to be candidate target of miR146a by four algorithms. Luciferase gene assay showed that miR146a also exerted down-regulatory effect on IFIT3translation by binding to3’-UTR of IFff3mRNA via a sequence-dependent manner in hepatoma cells.The basic level of1F1T3was also lower in HepG2.2.15cells than that in HepG2cells. Luciferase assay demonstrated that IF1T3translation could be partly recovered by miR146a inhibition in HepG2.2.15cells.7. miR146a repressed the induction of type Ⅰ IFN by5’ppp-RNA stimulation in hepatoma cellsmiR146a pre-silencing increased the induction fold of type I IFN after5"ppp-RNA transfection in HepG2.2.15cells, and IFIT3over-expression could achieve similar phenomenon.Luciferase assay demonstrated that TRAF6. another miR146a target and key adaptor for R1G-I signaling, was also regulated at translational level by miR146a via a "mis-match" paired interaction in hepatoma cells.Furthermore, IFIT3may inhibit HBV transcription directly either in an unknown pathway.Conclusion 1. miR146a expression in HBV-carried hepatoma cell line, HepG2.2.15, was higher than that in HepG2cells, which due to enhanced activity of miR146a promoter.2. miR146a promoted HBV life cycle both in vitro and in vivo, which was independent on the direct interaction between miR146a and HBV RNA.3. miR146a negatively regulated type I IFN response by targeting STAT1in hepatoma cells, and silencing miR146a would increase anti-HBV effect of type I IFN both in vitro and in vivo.4. Human IFIT3was a new target of miR146a, and could probably inhibit HBV directly.5. miR146a also negatively regulated5’ppp-RNA-dependent type I IFN induction in hepatoma cells by targeting IFIT3and TRAF6. Also, inhibition of miR146a would recover impaired intracellular innate immunity and augment type I IFN expression in hepatoma cells.Our findings demonstrated that miR146a up-regulation was one of the important epigenetic characteristics of hepatocyte during HBV infection; miR146a would attenuate type I IFN production and play roles via targeting essential signaling factors, including STAT1, IFIT3and TRAF6, which facilitated HBV life cycle; silencing miR146a could reverse HBV-induced immune inhibitory status in liver and expedite HBV clearance, which may be a promising strategy to design new drugs that can break "IFN-resistance".