Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Proc Natl Acad Sci U S A. 2012 Apr 10;109(15):5826-31. doi: 10.1073/pnas.1118118109. Epub 2012 Mar 26.
Human inducible nitric oxide synthase (hiNOS) gene expression is regulated by transcriptional and posttranscriptional mechanisms. The purpose of this study was to determine whether specific microRNA (miRNA) directly regulate hiNOS gene expression. Sequence analysis of the 496-bp hiNOS 3'-untranslated region (3'-UTR) revealed five putative miR-939 binding sites. The hiNOS 3'-UTR conferred significant posttranscriptional blockade of luciferase activity in human A549, HCT8, and HeLa cells. The hiNOS 3'-UTR also exerted basal and cytokine-stimulated posttranscriptional repression in an orientation-dependent manner. Functional studies demonstrated that transfection of miR-939 into primary human hepatocytes (HCs) significantly inhibited cytokine-induced NO synthesis in a dose-dependent manner that was abrogated by a specific miR-939 inhibitor. MiR-939 (but not other miRNAs) abolished cytokine-stimulated hiNOS protein in human HC, but had no effect on hiNOS mRNA levels. Site-directed mutagenesis of miR-939 bindings sites at +99 or +112 bp in the hiNOS 3'-UTR increased reporter gene expression. Furthermore, intact miR-939 binding sites at +99 or +112 positions were required for posttranscriptional suppression by miR-939. Cytokine stimulation directly increased miR-939 levels in human HC. Transfection of miR-939 inhibitor (antisense miR-939) enhanced cytokine-induced hiNOS protein and increased NO synthesis in vitro in human HC. Finally, cytokine or LPS injection in vivo in mice increased hepatic miR-939 levels. Taken together, these data identify that miR-939 directly regulates hiNOS gene expression by binding in the 3'-UTR to produce a translational blockade. These findings suggest dual regulation of iNOS gene expression where cytokines induce iNOS transcription and also increase miR-939, leading to translational inhibition in a check-and-balance system.
人类诱导型一氧化氮合酶（hiNOS）基因表达受转录和转录后机制调控。本研究旨在确定特定的 microRNA（miRNA）是否直接调节 hiNOS 基因表达。对 496bp 的 hiNOS 3'-非翻译区（3'-UTR）的序列分析显示，有五个可能的 miR-939 结合位点。hiNOS 3'-UTR 在人 A549、HCT8 和 HeLa 细胞中显著阻断了荧光素酶活性的转录后表达。hiNOS 3'-UTR 还以依赖于方向的方式发挥基本和细胞因子刺激的转录后抑制作用。功能研究表明，miR-939 转染原代人肝细胞（HCs）可显著抑制细胞因子诱导的 NO 合成，且这种抑制作用可被特异性 miR-939 抑制剂所消除。miR-939（而非其他 miRNA）消除了人 HC 中细胞因子刺激的 hiNOS 蛋白，但对 hiNOS mRNA 水平没有影响。在 hiNOS 3'-UTR 的+99 或+112 位碱基处进行 miR-939 结合位点的定点突变，可增加报告基因的表达。此外，miR-939 结合位点在+99 或+112 位的完整性对于 miR-939 的转录后抑制是必需的。细胞因子刺激可直接增加人 HC 中的 miR-939 水平。miR-939 抑制剂（反义 miR-939）的转染增强了人 HC 中细胞因子诱导的 hiNOS 蛋白，并增加了体外的 NO 合成。最后，体内注射细胞因子或 LPS 可增加小鼠肝组织中的 miR-939 水平。综上所述，这些数据表明，miR-939 通过与 3'-UTR 结合直接调节 hiNOS 基因表达，从而产生翻译阻断。这些发现表明 iNOS 基因表达受到双重调控，其中细胞因子诱导 iNOS 转录，并增加 miR-939，导致在一个检查和平衡系统中产生翻译抑制。
