Regulation of NADPH Oxidase Activity Is Associated with miRNA-25-Mediated NOX4 Expression in Experimental Diabetic Nephropathy
Regulation of NADPH Oxidase Activity Is Associated with miRNA-25-Mediated NOX4 Expression in Experimental Diabetic Nephropathy
复制标题
实验性糖尿病肾病中 NADPH 氧化酶活性的调节与 miRNA-25 介导的 NOX4 表达相关
DOI：
10.1159/000322105
复制
发表时间：
2010-01-01
影响因子：
4.2
通讯作者：
Yi, Fan
中科院分区：
文献类型：
--
作者：
Fu, Yibing;Zhang, Yan;Yi, Fan
背景/目的：尽管已有大量研究探讨了糖尿病肾病(DN)中NADPH氧化酶活性的调节机制，但有关microRNAs(MiRNAs)对NADPH氧化酶表达的调节作用的信息却很少。因此，本研究旨在检测miRNAs是否在高血糖状态下对NADPH氧化酶的主要催化亚基NOX4的表达起到重要的调节作用。方法：用链脲佐菌素复制糖尿病大鼠模型。本研究采用miRNA芯片、Western印迹、实时定量RT-PCR和荧光素酶报告分析等方法。结果：在预测与大鼠NOX4结合的5个miRNAs中，糖尿病大鼠肾脏和高糖处理的系膜细胞中miRNA-25水平均显著降低，同时NOX4表达水平升高。在体外研究中，我们发现，在miRNA-25抑制剂转染组中，NADPH活性增加了226.2%，而在miRNA-25前体转染组中，其活性降低了51.0%。MIR-25抑制剂显著增加了NOX4的mRNA和蛋白水平。然后，我们通过荧光素酶报告基因分析表明，miR-25通过直接靶向3‘-UTR来负调控NOX4的表达。结果表明，转染miR-25前体后，NOX4 3‘-UTR的荧光素酶活性下降了39.5%，而突变序列恢复到了79.4%。最后，我们的结果表明miR-25介导的NOX4mRNA水平可能是通过调节mRNA的稳定性来实现的。结论：首次发现miRNA-25可能作为内源性基因沉默因子，参与调控NOX4在糖尿病肾病中的表达和功能。版权所有(C)2010 S.Karger AG，巴塞尔。
Background/Aims: Although numerous studies have explored the mechanisms regulating the enzyme activity of NADPH oxidase in diabetic nephropathy (DN), little information is available for the contribution of microRNAs (miRNAs) to the regulation of NADPH oxidase expression. Therefore, the present study was to test whether miRNAs importantly contribute to the regulation of NOX4 expression, a major catalytic subunit of NADPH oxidase under hyperglycemia. Methods: Diabetic rats were induced by streptozotocin. miRNA microarray, Western blot, real-time RT-PCR and luciferase reporter assays were employed in this study. Results: Among 5 miRNAs, which are predicted to have a binding capacity to rat NOX4, the miRNA-25 level was significantly reduced both in the kidney from diabetic rats and in high glucose-treated mesangial cells, accompanied by the increases in NOX4 expression levels. In an in vitrostudy, we found that NADPH activity was increased by 226.2% in miRNA-25 inhibitor transfected cells and decreased by 51.0% in miRNA-25 precursor transfected cells. miR-25 inhibitor dramatically increased both NOX4 mRNA and protein levels. We then showed that miR-25 negatively regulated NOX4 expression by directly targeting the 3'-UTR by luciferase reporter assays. It was found that transfection of miR-25 precursor significantly decreased the luciferase activity of NOX4 3'-UTR by 39.5%, whereas the mutant sequence restored levels to 79.4%. Finally, our results indicated that the miR-25-mediated NOX4 mRNA level may result from the regulation of mRNA stability. Conclusions: These findings for the first time indicate that miRNA-25 may serve as an endogenous gene silencing factor and contributes to the regulation of NOX4 expression and function in DN. Copyright (C) 2010 S. Karger AG, Basel.