Abstract
We previously identified that miR-130a downregulates HCV replication through two independent pathways: restoration of host immune responses and regulation of pyruvate metabolism. In this study, we further sought to explore host antiviral target genes regulated by miR-130a. We performed a RT² Profiler™ PCR array to identify the host antiviral genes regulated by miR-130a. The putative binding sites between miR-130a and its downregulated genes were predicted by miRanda. miR-130a and predicted target genes were over-expressed or knocked down by siRNA or CRISPR/Cas9 gRNA. Selected gene mRNAs and their proteins, together with HCV replication in JFH1 HCV-infected Huh7.5.1 cells were monitored by qRT-PCR and Western blot. We identified 32 genes that were significantly differentially expressed more than 1.5-fold following miR-130a overexpression, 28 of which were upregulated and 4 downregulated. We found that ATG5, a target gene for miR-130a, significantly upregulated HCV replication and downregulated interferon stimulated gene expression. miR-130a downregulated ATG5 expression and its conjugation complex with ATG12. ATG5 and ATG5-ATG12 complex affected interferon stimulated gene (ISG) such as MX1 and OAS3 expression and subsequently HCV replication. We concluded that miR-130a regulates host antiviral response and HCV replication through targeting ATG5 via the ATG5-dependent autophagy pathway.
Highlights
Noncoding RNA plays an important role in viral infection and host immune responses [1].MicroRNA is a class of small non-coding RNA that negatively regulates gene expression atCells 2019, 8, 338; doi:10.3390/cells8040338 www.mdpi.com/journal/cellsCells 2019, 8, 338 the transcriptional or post-transcriptional level
Autophagy has been shown to be required for hepatitis C virus replication initiation, the downregulation of autophagy molecules is associated with reduced HCV replication [19]
We conclude that miR-130a plays an important role in regulating HCV replication through targeting autophagy pathway genes, in addition to its previously identified role in regulating metabolism via PKLR
Summary
Noncoding RNA plays an important role in viral infection and host immune responses [1].MicroRNA (miRNA) is a class of small non-coding RNA that negatively regulates gene expression atCells 2019, 8, 338; doi:10.3390/cells8040338 www.mdpi.com/journal/cellsCells 2019, 8, 338 the transcriptional or post-transcriptional level. HCV replication through the expression of several host innate immunity related genes including type I. We found that autophagy 5 (ATG5) expression was one of the most significantly downregulated genes in the context of miR-130a overexpression. HCV infection activates the JAK-STAT pathway leading to the production of several hundred interferon stimulated genes (ISGs) that produce an antiviral state to restrict HCV spread within liver [1,3,13,14,15,16,17]. The ATG5-ATG12 complex negatively regulates type I interferon (IFN) production in mouse embryonic fibroblasts (MEFs) to facilitate vesicular stomatitis virus replication [24]. ATG5 has been shown to be essential for type I IFN production in plasmacytoid dendritic cells infected with vesicular stomatitis virus [25].
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