MicroRNA-196 represses Bach1 protein and hepatitis C virus gene expression in human hepatoma cells expressing hepatitis C viral proteins.
MicroRNA-196 represses Bach1 protein and hepatitis C virus gene expression in human hepatoma cells expressing hepatitis C viral proteins.
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DOI：
10.1002/hep.23401
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发表时间：
2010-05
期刊：
影响因子：
13.5
通讯作者：
Bonkovsky, Herbert L.
中科院分区：
文献类型：
--
作者：
Hou, Weihong;Tian, Qing;Zheng, Jianyu;Bonkovsky, Herbert L.
丙型肝炎病毒(丙型肝炎病毒)直接引起氧化应激和肝损伤。Bach1是一种拉链(BZip)哺乳动物转录抑制因子，它负性调节血红素加氧酶1(Hmox1)，这是一种具有抗氧化和抗炎活性的关键细胞保护酶。MicroRNAs是一种小的非编码RNA(~22nt)，是基因表达的重要调节因子。MicroRNAs是否以及如何调控Bach1或丙型肝炎病毒在很大程度上是未知的。本研究的目的是确定miR-196是否调节Bach1、Hmox1和/或丙型肝炎病毒的基因表达。本研究使用的是Con1株的复制子细胞系(Con1和9-13)以及基于J6/JFH1的丙型肝炎病毒细胞培养系统。用qRT-PCR和Western blots检测miR-196模拟物对Bach1、Hmox1和丙型肝炎病毒RNA和蛋白水平的影响。用双Glo™荧光素酶检测系统测定报告基因活性。MIR-196模拟显著下调Bach1和上调Hmox1基因的表达，并抑制丙型肝炎病毒的表达。双重荧光素酶报告基因检测结果表明，miR-196模拟物可显著降低突变株Bach13‘-UTR依赖的荧光素酶活性，但对突变株Bach13’-UTR依赖的荧光素酶活性无明显影响。此外，突变的miR-196对依赖于Bach13‘-UTR的荧光素酶活性没有明显的影响。MIR-196直接作用于Bach1mRNA的3‘-UTR，并在翻译上抑制该蛋白的表达，上调Hmox1的表达。MIR-196还可抑制丙型肝炎病毒复制子细胞系(1b型)和J6/JFH1(2a型)丙型肝炎病毒细胞培养系统中丙型肝炎病毒的表达。因此，miR-196在人肝细胞Hmox1/Bach1的表达和丙型肝炎病毒的表达调控中发挥作用。MiR-196的过表达有望成为预防或改善丙型肝炎感染和保护慢性丙型肝炎肝损伤的潜在新策略。
Hepatitis C virus (HCV) directly induces oxidative stress and liver injury. Bach1, a zipper (bZip) mammalian transcriptional repressor, negatively regulates heme oxygenase 1 (HMOX1), a key cytoprotective enzyme that has anti-oxidant and anti-inflammatory activities. microRNAs are small non-coding RNAs (~22 nt) that are important regulators of gene expression. Whether and how microRNAs regulate Bach1 or HCV are largely unknown. The aims of this study were to determine whether miR-196 regulates Bach1, HMOX1, and/or HCV gene expression. HCV replicon cell lines (Con1 and 9-13) of the Con1 isolate and J6/JFH1-based HCV cell culture system were used in this study. The effects of miR-196 mimic on Bach1, HMOX1 and HCV RNA and protein levels were measured by qRT-PCR and Western blots, respectively. The Dual Glo™ Luciferase Assay System was used to determine reporter activities. miR-196 mimic significantly down-regulated Bach1 and up-regulated HMOX1 gene expression, and inhibited HCV expression. Dual luciferase reporter assays demonstrated that transfection of miR-196 mimic resulted in a significant decrease in Bach1 3′-UTR-dependent luciferase activity but not in mutant Bach1 3′-UTR-dependent luciferase activity. Moreover, there was no detectable effect of mutant miR-196 on Bach1 3′-UTR-dependent luciferase activity. miR-196 directly acts on the 3′-UTR of Bach1 mRNA and translationally represses the expression of this protein, and up-regulates HMOX1. miR-196 also inhibits HCV expression in HCV replicon cell lines (genotype 1b) and in J6/JFH1 (genotype 2a) HCV cell culture system. Thus, miR-196 plays a role in both HMOX1/Bach1 expression and the regulation of HCV expression in human hepatocytes. Over-expression of miR-196 holds promise as a potential novel strategy to prevent or ameliorate hepatitis C infection, and to protect against liver injury in chronic HCV infection.
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