| Background and objective: Hepatitis C virus(HCV) is a hepatotropic RNA virus of the genus Hepacivirus in the Flaviviridae family. HCV has become a globally spread virus, leading to a global health problem which requires significant resources for its prevention and control. HCV can cause acute and chronic hepatitis in humans and chimpanzees, if untreated it may progress to cirrhosis and hepatocellular carcinoma. HCV is a positive-sense, single-stranded enveloped RNA virus approximately 9600 nucleotides in length.MicroRNAs(miRNA) are 19-24 nucleotides long, noncoding RNA molecules that repress gene expression post-transcriptionally. Mi RNAs play very important roles in maintaining normal human body physiology conditions, and abnormal mi RNA expressions have been found related to many human diseases spanning from psychiatric disordersto malignant cancers. Moreover, they play a major role in regulating hostgene expression in virus infected cells and cancers caused by virus infection. Mi R-122 is a liver specific microRNA and is the microRNA most abundantly expressed in the liver. There are reports that mi R-122 is involved in maintaining the normal function of the liver. Dramaticall y, miR-122 was used by HCV for triggering virus replication by repression of the heme oxygenase-1 or by interaction with the 5’ UTR of HCV RNA. And mi R-122 was found to be dysregulated in different stage of HCV infected liver tissues and serum.Signal transducer and activator of transcription factors(STATs) are a family of transcription factors that plays essential roles in cell growth, development, proliferation, immune defense and differentiation. Seven members of the STAT family have been identified and characterized, including STAT1, STAT2, STAT3, STAT4, STAT5 a, STAT5 b and STAT6. Among the STAT family, STAT3 has been identified as a critical regulator in tumor cells. The activation of STAT3 might be one of the mechanisms by which Human papilloma virus(HPV) achieve oncogenic transformation. E2 as an early protein of HPV 16, potentiates TNF-αmediated NF-κB activation through activation of STAT3 in the presence of TNF-α. And this resulted in the reversal of E2-induced apoptosis, which leading to cell survial. Robert S. Tacke et al found that extracellular hepatitis C virus core protein activates STAT3 in human monocytes, macrophages and dendritic cells through an IL-6 autocrine pathway. Previous studies pointed that STAT3 act as a negative regulator of typ e I IFN-mediated response.Non-coding RNAs(ncRNAs) are transcribed RNA molecules with little or non-protein coding capacity. Among all types of ncRNAs, long non-coding RNAs(Lnc RNAs), a regulatory ncRNAs, play critical roles in almost every aspect of cellular processes. NEAT1 is one of several LncRNAs whose expression is changed by HIV-1 infection, and the knockdown of NEAT1 enhanced HIV-1 production through increased nucleus-to-cytoplasm export of Rev-dependent instability element(INS)-containing HIV-1 m RNAs. Apart from HIV, according to a large number of recent studies, LncRNAs are related to the development and progress of HCV which caused hepatocellular carcinoma(HCC). Emerging evidences point to the involvement of LncRNAs in many aspects of the immune response, including several pathways related to innate immunity. Most recently, IFN-induced LncRNA-CAPK2 has an inhibitory effort on transcription of IFN-stimulated antiviral genes, which suggest that CMPK2 acts as a negative regulator of IFN response. Despite these discoveries, the role of Lnc RNAs in key aspects of the immune response including the nearly ubiquitous and functionally crucial interferon(IFN) response remains to further investigation.The present study demonstrated that miR-122 inhibited the expression of signal transducer and activator of transcription 3(STAT3), an antivirus response repressor. Interestingly, HCV RNA acted as a “microRNA sponge” which can up-regulate STAT3 expression by sealing mi R-122. Subsequently, we confirmed that this mi R-122 sponge function of HCV RNA can repress poly I: C stimulated type I IFN expression. Furthermore, we identify that several LncRNAs can be up-regulate and down-regulated by phosphorylated STAT3 by using human PCR array method. And among these LncRNAs, Lnc-Lethe was involved in the HCV replication. Transfection of si RNA Lethe partially blocked the replication of HCV in Huh-7 cells. Data also indicated that when transfected with si RNA Lethe, the expression levels of PKR, OAS and IRF1, which were all ISGs, were all up regulated. Based on our findings from Lethe knockdown, we have identified that Lethe, which was upregulated by activated STAT3, may promoting the replication of HCV through a negative regulatory mechanism of type I IFN response.This study provided a further understanding of the complicated roles of mi R-122 in the progress of HCV virus infection, and supported the miR-122 inhibition strategy in anti-HCV infection treatment. In summary, these findings would be helpful to further understand the molecular mechanism of HCV replication and the influence of STAT3 on HCV replication. Also we have done a preliminary study about the mechanism of how Lethe works. Our future analysis will focus on whether Lethe would be potential diagnostic even therapeutic targets for HCV infection.Materials and methods:1. Cell cultureHuh-7 cells were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum, 100 IU/ml penicillin and 10 mg/m L streptomycin. All cells were maintained at 37℃ under an atmosphere of 5% CO2.2. Real-time reverse transcriptase quantitative PCRQuantitive RT-PCR analysis was used to determine the relative expression level of miR-122,HCV RNA,Lnc RNA-Letheand ISGs m RNA. Total RNA was extracted from tissues using Trizol according to the manufacturer’s instructions. The expression level of mRNA was detected by TaqMan mi RNA RT-Real Time PCR.3. 3′-UTR and 5′-UTR Luciferase Reporter AssaysTo generate 3′-UTR luciferase reporter, a segment of 567 bp 3′-UTR from STAT3 was cloned into the downstream of the firefly luciferase gene in p GL3-Control Vector(Promega). Mi R-122 mimic and miR-122 inhibitor were synthesized. For luciferase reporter assays, Huh-7 cells were seeded in 48-well plates. Luciferase reporter vectors were co-transfected with mi R-122 mimic or inhibitor by using lipofectamine 2000. Two days later, cells were harvested and assayed with the Dual-Luciferase Assay. The results were expressed as relative luciferase activity.To generate 5′-UTR luciferase reporter, wild type or mutant HCV RNA 5′-UTR was cloned into the upstream of the firefly luciferase gene in pGL3-Basic vector.Mi R-122 mimic andpGL3-Basic-HCV5′-UTR vectors were co-transfected into HEK293 T cells, p RL-TK vector was used for data normalization.4. Western blottingWestern blotting was used to determine the relative expression level of STAT3, HCVNS5 A,NS3 and ISGs Protein. Protein extracts were boiled in SDS/β-mercaptoethanol sample buffer, and 20 μg samples were loaded into each lane of 10% polyacrylamide gels. The proteins were separated by electrophoresis, and the proteins in the gels were blotted onto PVDF membranes by electrophoretic transfer.5. Lnc RNAs PCR arrayThe Lnc RNA PCR array method was used to detect the different expression levels of LncRNAs in STAT3 activated and control cells. The sample preparation and Human LncRNA PCR Array Kit were prepared based on the manufacturer’s protocols. Purified RNA was reversed transcribed into c DNA with Human LncRNA Profiler c DNA synthesis Buffer. Real-time PCR and PCR array amplification was conducted by IQ5 machine(Bio-Rad) and the conditions were set as follows: 15 seconds at 95 °C, followed by 40 cycles of: 15 seconds at 95 °C, 30 seconds at 65 °C and 30 seconds at 72 °C. Subsequently a melt curve analysis was performed to determine the specificity of the PCR reaction. The relative gene expression was calculated by using △△Ct method.6. siRNA TransfectionMonolayers of Huh-7 cells with 70% confluence in 6 well plates were transfected with siRNA Lethe or with si RNA negative control. Transfection was performed using Lipofectamine? 2000 according to the manufacturer’s instructions. In brief, Huh-7 cells(in serum free DMEM) were transfected using different amount of Lipofectamine? 2000(Invitrogen) according to si RNA quantity. Eight hours postinfection, the cells were harvested to measure the viral gene m RNA level by real-time PCR. Negative control sequence was a missense sequence.7. Statistical analysisData were analyzed by using SPSS Statistical Package version 16. Independent two group’s analyses are used t-test. P < 0.05 was considered statistically significant.Results:1. Mi R-122 repressed STAT3 expression by targeting STAT3 mRNA 3′-UTRWe searched new target genes by using bioinformatics tools. Predicted by using online bioinformatics tool: TargetScan,we found that STAT3 mRNA may be target by mi R-122 3′-UTR directly.To validate whether STAT3 is indeed the target gene of mi R-122, a 567 bp segment of STAT3 3′-UTR containing predicted mi R-122 binding site was cloned into the downstream of the firefly luciferase reporter gene in the pGL3 control vector(designated as p GL3-STAT3) for the dual luciferase assay. Huh-7cells were co-transfected with p GL3-STAT3 and miR-122 mimics or inhibitor.Compared with the mi RNA control, the luciferase activity was significantly suppressed by the miR-122, about 42.1%(P<0.05). Furthermore, the luciferase activity was significantly up-regulated by the miR-122 inhibitor compared with the anti-mi R control, about 38.4%(P<0.05).These results indicate that miR-122 targets the 3′-UTR of STAT3, leading to the change of firefly luciferase translation.Seed sequence mutation clone was also used to further confirm the binding site for miR-122. The vector contains putative mi R-122 binding region in the 3′-UTR of STAT3 with 5 mutant nucleotides(designated as pGL3-STAT3-Mu) was used and wild type STAT3 vector was used as control. The enzyme activity was reduced about 56.2% in cells transfected with p GL3-STAT3 compared with pGL3-STAT3-Mu(P<0.05). These data indicate that miR-122 may suppress STAT3 expression through binding to seed sequence at the 3′-UTR of STAT3, and STAT3 may be a direct target gene of miR-122.2. HCV RNA overexpression sponged mi R-122 and rescued STAT3 expression in Huh-7 cellsTo explore whether HCV RNA can protect mi R-122 target genes expression by interact with mi R-122, we first repeated the experiment that miR-122 can target HCV mRNA 5′-UTR. MiR-122 overexpression can enhance luciferase expression compared with scramble miR-control. When three nucleotides of mi R-122 target sites were changed, the relative firefly luciferase activity was significantly reduced. Transient transfec ted the vector encode 9658 bp HCV genotype 1b RNA into Huh-7 cells and qRT-PCR was used to determine the expression of mi R-122 24 hours after transfection. We found that miR-122 expression reduced slightly compared with control, but the difference is not si gnificant. Subsequently, STAT3 expression was up-regulated by overexpressed HCV RNA compared with empty vector and miR-122 target sites mutant vector which means HCV RNA can protect STAT3 expression by absorbing mi R-122.3. HCV RNA overexpression inhibit IFN expression by rescuing STAT3STAT3 is considered to be a negatively regulator oftype I IFN-mediated antiviral response. STAT3 knockdown or knockout cells displayed enhanced gene expression and antiviralactivity in response to IFN-α and IFN-β. To explore the response of HCV RNA overexpression to I type IFN generation, we detect IFN-α and IFN-β expression by q RT-PCR at four time points after plasmid and polyI: C transfection. We found that HCV RNA can repress IFN-α and IFN-β expression but mi R-122 target sites mutant HCV RNA cannot which means HCV RNA can repress cell anti-virus response by absorbing mi R-122 directly.4. Activation of STAT3 Increases HCV ReplicationSTAT3 activation requires posttranslational modifications through phosphorylation at tyrosine 705(Y705) to be functionally active. To investigate if STAT3 activation affects the HCV life cycle, we activated STAT3 by way of exogenous cytokine treatment with LIF, a known activator of STAT3. As expected, the levels of STAT3-Y705 were significantly increased with the treatment of LIF in a time dependent manner and dose dependent manner, while the total STAT3 levels were not changed with the treatment of LIF. When Huh-7 cells pretreated with LIF for a time course of 0-60 minutes before infection of HCV, the m RNA levels of HCV increased in a time dependent manner. And when treated Huh 7 cells with different doses of LIF prior to infection of HCV, the mRNA levels of HCV increased in a dose dependent manner. Consistent with this increase in HCV mRNA levels, we observed a marked increase in NS5 A and NS3 protein levels with LIF treatment prior to infection of HCV JFH-1 strain. These results indicate that activated STAT3 plays an important role in HCV replication or potentially induce the expression of specif ic genes that are in turn able to create an environment that is favorable for HCV replication.5. Activation of STAT3 regulate LncRNAsThe Lnc RNA PCR array method was used to detect the different expression levels of LncRNAs in STAT3 activated and control cells. 10 LncRNAs were upregulated and 6 LncRNAs were downregulated in STAT3 activated Huh-7 cells. These identifications were based on the criterion that all changes were at least 2-fold or more compared to control cells. Among all these upregulated or downregulated Lnc RNAs, we then further studied the characterization of Lethe as it was upregulated highest level in all the LncRNAs that were upregulated by STAT3 activation.To confirm and extend this observation we compared the m RNA levels of Lethe in Huh-7 cells treated with LIF or AG490 with control cells and verified that expression level of Lethe in the LIF treated cells was indeed upregulated by approximately 5 fold and in the AG490 treated cells was decreased. The mRNA levels of Lethe in the HCV infected cells was upregulated by almost 4 fold Compared with control cells, which has similar effect as pretreatment with LIF prior to infection of HCV.6. Depletion of Lethe inhibit HCV replicationThe above data show that HCV infection increases the expression level of Lethe, but we wondered how Lethe expression might reciprocally influence HCV replication. Then we employed small interfering RNA(siRNA) knockdown of Lethe. Three pairs of Lethe siRNA and a control si RNA were transfected into Huh-7 cells, and si- Lethe-3 can reduce the RNA levels approximately 80% compared to control si RNA. Then Huh-7 cells were transfected with si- Lethe-3 or a control siRNA, and infected with HCV JFH-1. The knockdown of Lethe decreased HCV m RNA levels more than 50%. The Western blot assay showed that NS5 A and NS3 protein levels were both decreased more than 50% with the knockdown of Lethe. The HCV m RNA levels would not increase in Huh-7 cells transfected with Lethe si RNA after the treatment of LIF compared with control cells. These results indicate that Lethe promotes HCV replication.7. Lethe may promote HCV replication by regulating type I IFN ResponseAs previous study shows that STAT3 is a negative regulator of type I IFN response, we wondered whether the Lethe, upregulated by activated STAT3, would affect type I IFN response.Then the effects of decreasing Lethe expression on the expression of ISGs, including PKR, OAS and IRF1 were investigated. It is shown that the exp ression levels of PKR, OAS and IRF1 were all upregulated by transfected with Lethe si RNA.Then the real-time PCR assay also confirmed that Lethe si RNA would upregulate the m RNA levels of PKR, OAS and IRF1. To confirm and extend this observation we investiga ted the m RNA levels of PKR, OAS and IRF1 when Huh-7 cells treated with LIF and then transfected with Lethe siRNA. And as expected the m RNA levels of all three ISGs were much higher than those in Huh-7 cells treated with LIF and control siRNA or LIF only. Together, these results suggest that Lethe may promote HCV replication by regulating type I IFN Response.Conclusions:1. Hepatitis C virus represses cellular anti-virus response by up-regulating STAT3 expression through sponging mi R-1222. Activation of STAT3 up regulated Lnc-Lethe which promoted HCV replication in Huh-7 cells.3. Lnc-Lethe may promote HCV replication by regulating typeⅠIFN response. |
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