Liver-Biliary-Pancreatic Center and the Liver, Digestive Diseases, and Metabolism Laboratory, Carolinas Medical Center, Charlotte, NC 28232-2861, USA.
Hepatology. 2010 May;51(5):1494-504. doi: 10.1002/hep.23401.
Hepatitis C virus (HCV) directly induces oxidative stress and liver injury. Bach1, a basic leucine zipper mammalian transcriptional repressor, negatively regulates heme oxygenase 1 (HMOX1), a key cytoprotective enzyme that has antioxidant and anti-inflammatory activities. microRNAs (miRNAs) are small noncoding RNAs ( approximately 22 nt) that are important regulators of gene expression. Whether and how miRNAs 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 way of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, 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'-untranslated region (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 messenger RNA 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. Overexpression 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.
丙型肝炎病毒（HCV）直接诱导氧化应激和肝损伤。Bach1 是一种基本亮氨酸拉链哺乳动物转录抑制剂，可负调控血红素加氧酶 1（HMOX1），后者是一种具有抗氧化和抗炎作用的关键细胞保护酶。microRNAs（miRNAs）是约 22 个核苷酸的小非编码 RNA，是基因表达的重要调节剂。miRNAs 是否以及如何调节 Bach1 或 HCV 在很大程度上尚不清楚。本研究旨在确定 miR-196 是否调节 Bach1、HMOX1 和/或 HCV 基因表达。本研究使用了来自 Con1 分离株的 HCV 复制子细胞系（Con1 和 9-13）和 J6/JFH1 基于的 HCV 细胞培养系统。通过定量实时聚合酶链反应（qRT-PCR）和 Western blot 分别测量 miR-196 模拟物对 Bach1、HMOX1 和 HCV RNA 和蛋白质水平的影响。双荧光素酶报告基因检测系统用于测定报告基因活性。miR-196 模拟物显著下调 Bach1 并上调 HMOX1 基因表达，抑制 HCV 表达。双荧光素酶报告基因检测显示，miR-196 模拟物转染导致 Bach1 3'-非翻译区（UTR）依赖性荧光素酶活性显著降低，但突变 Bach1 3'-UTR 依赖性荧光素酶活性没有检测到。此外，突变 miR-196 对 Bach1 3'-UTR 依赖性荧光素酶活性没有检测到影响。
miR-196 直接作用于 Bach1 mRNA 的 3'-UTR，翻译抑制该蛋白的表达，并上调 HMOX1。miR-196 还抑制 HCV 复制子细胞系（基因型 1b）和 J6/JFH1（基因型 2a）HCV 细胞培养系统中的表达。因此，miR-196 在人类肝细胞中既发挥调节 HMOX1/Bach1 表达的作用，也发挥调节 HCV 表达的作用。miR-196 的过表达有望成为预防或改善丙型肝炎感染以及预防慢性 HCV 感染所致肝损伤的潜在新策略。
