| Background:HCV and HBV infection have been shown to regulate miR-130a expression both in infected patient biopsies and in infected cultured cells.However,the detailed mechanism by which miR-130a regulates HCV and HBV replication is not well understood.Aims:We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and HBV replication.Methods:We investigated the effect of miR-130a overexpression,as well as the addictive effect of miR-130a and Calcitriol,on HCV replication and type Ⅰ interferon pathway in Con lb replicon and in JFH1 infected Huh7.5.1 cells.We used bioinformatics software including miRanda,TargetScan,PITA and RNAhybrid to predict potential miR-130a target genes.To validate the target genes,dual-luciferase reporter gene assays were performed.miR-130a and its target genes were overexpressed,or knocked down by siRNA or by CRISPR/Cas9 gRNA,respectively.Selected gene mRNAs and their proteins,together with HCV replication in OR6 cells,JFH1 HCV-infected Huh7.5.1 cells and JFH1 HCV-infected human primary human hepatocytes(PHHs),and HBV replication in HepAD38 cells,HBV-infected NTCP-Huh7.5.1 cells and HBV-infected NTCP-PHHs were monitored by qRT-PCR and Western blot,respectively.Results:We found that miR-130a overexpression significantly inhibits HCV replication both in HCV replicon and in JFH1-infected Huh7.5.1 cells.Over expression of miR-130a upregulated the expression of type Ⅰ IFN(IFNα/IFNβ)and some interferon stimulated genes,such as CH25H and Mxl,which are involved in innate immune response.We confirmed that the upregulation of ISGs stimulated by miR-130a depends on intact type-ⅠIFN signaling.Calcitriol potentiates the anti-HCV effect of miR-130a in both Conlb replicon and JFH1 cells.Intriguingly,this potentiating effect of calcitriol on miR-130a was not through upregulating the expression of cellular miR-130a or through increasing the miR-130a-mediated IFNα/β production,but probably through inhibiting the expression of LDLR to inhibit HCV entry.We identified 2152 potential miR-130a target genes by bioinformatics analysis.We confirmed 110 genes whose expression was related to viral pathogenesis and immunity by qPCR.Of these,pyruvate kinase in liver and red blood cell(PKLR),low-density lipoprotein receptor(LDLR)and IL18 Binding protein(IL18BP)were found to be regulated by miR-130a overexpression.Dual luciferase reporter assay specifically confirmed that miR-130a targets PKLR.We found that miR-130a overexpression(miR-130a mimic)knocked down PKLR mRNA and protein levels in OR6 cells,Huh7.5.1 cells,HepAD38 cells,and PHHs.miR-130a inhibitor and gRNA increased PKLR expression.miR-130a mimic and PKLR siRNA or gRNA knockdown inhibited pyruvate production,HCV replication in JFH1-infected Huh7.5.1 cells and JFH1-infected PHHs,and HBV replication in HepAD38 cells,HBV infected NTCP-Huh7.5.1 cells and NTCP-PHHs,while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication.Supplemental pyruvate increased HCV and HBV replication and rescued HCV and HBV replication inhibition by miR-130a mimic and PKLR knockdown.Moreover,miR-130a levels were not affected by PKLR overexpression,knock down or supplemental pyruvate.Conclusion:miR-130a regulates HCV and HBV production through restoring type I interferon pathway and through its targeting of PKLR and its subsequent pyruvate production.Pyruvate is a pivotal intermediate molecule in energy supply and metabolic building block for cells and virus.Here we show that miR-130a play critical role in both immunity response and cell metabolism.Our data provides novel insights into understanding miR-130a regulated type I IFN pathway and key metabolic enzymatic pathway steps,which are subverted by HCV and HBV replication.Understanding immune control of key metabolic enzymatic steps in infection will promote the future development of novel therapeutic modalities based on metabolic modifiers that either enhance protection or inhibit infection. |
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