Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu 610052, China.
Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
Cells. 2019 Apr 10;8(4):338. doi: 10.3390/cells8040338.
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.
我们之前发现 miR-130a 通过两种独立的途径下调 HCV 复制：恢复宿主免疫反应和调节丙酮酸代谢。在这项研究中，我们进一步探索了 miR-130a 调节的宿主抗病毒靶基因。我们进行了 RT² Profiler™ PCR 阵列以鉴定受 miR-130a 调控的宿主抗病毒基因。miRanda 预测了 miR-130a 与其下调基因之间的假定结合位点。通过 siRNA 或 CRISPR/Cas9 gRNA 过表达或敲低 miR-130a 和预测的靶基因。通过 qRT-PCR 和 Western blot 监测 JFH1 HCV 感染的 Huh7.5.1 细胞中选定基因的 mRNA 和其蛋白以及 HCV 复制。miR-130a 过表达后，有 32 个基因的表达显著差异超过 1.5 倍，其中 28 个上调，4 个下调。我们发现，miR-130a 的靶基因 ATG5 显著上调 HCV 复制并下调干扰素刺激基因表达。miR-130a 下调 ATG5 表达及其与 ATG12 的缀合复合物。ATG5 和 ATG5-ATG12 复合物影响干扰素刺激基因 (ISG)，如 MX1 和 OAS3 的表达，进而影响 HCV 复制。我们得出结论，miR-130a 通过靶向 ATG5 并通过 ATG5 依赖性自噬途径调节宿主抗病毒反应和 HCV 复制。
