MicroRNA-146 Inhibits Thrombin-Induced NF-κB Activation and Subsequent Inflammatory Responses in Human Retinal Endothelial Cells
MicroRNA-146 Inhibits Thrombin-Induced NF-κB Activation and Subsequent Inflammatory Responses in Human Retinal Endothelial Cells
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DOI：
10.1167/iovs.13-13631
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发表时间：
2014-08-01
影响因子：
4.4
通讯作者：
Xu, Shunbin
中科院分区：
文献类型：
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作者：
Cowan, Colleen;Muraleedharan, Chithra K.;Xu, Shunbin
目的.核因子-κ B（NF-κ B B）是免疫和炎症反应的关键调节因子，在糖尿病视网膜病变（DR）等微血管并发症中起重要作用。凝血酶通过G蛋白偶联受体（GPCR）超家族成员蛋白酶激活受体（PAR）1激活NF-κ B，参与DR的发生。使用TargetScan算法进行目标预测。预测的靶标通过荧光素酶报告基因测定进行实验验证。用miRNA模拟物或antimiR转染人视网膜内皮细胞（HREC）并用凝血酶处理。通过定量（q）RT-PCR分析miR-146和相关蛋白编码基因的表达水平。通过白细胞粘附试验分析HREC的功能变化。我们发现，caspase募集结构域（CARD）的蛋白10（CARD 10），一个重要的支架/衔接蛋白的GPCR介导的NF-κ B活化途径，是一个直接的目标，miR-146。凝血酶处理导致HREC中NF-κ B依赖性的miR-146上调;而转染miR-146模拟物导致CARD 10显著下调并阻止凝血酶诱导的NF-κ B活化，表明miR-146通过靶向CARD 10对凝血酶诱导的NF-κ B负反馈调节。此外，miR-146的过表达阻止了凝血酶诱导的白细胞与HREC粘附的增加。我们发现了一种新的负反馈调节机制，凝血酶诱导的GPCR介导的NF-κ B激活的miR-146。结合我们先前报道的miR-146对RECs中IL-1 R/toll样受体（TLR）介导的NF-κ B激活的负反馈调节，我们的结果强调了miR-146对DR中多种NF-κ B激活途径和相关炎症过程的关键负调控作用。
PURPOSE. Nuclear factor-kappa B (NF-kappa B), a key regulator of immune and inflammatory responses, plays important roles in diabetes-induced microvascular complications including diabetic retinopathy (DR). Thrombin activates NF-kappa B through protease-activated receptor (PAR)-1, a member of the G-protein-coupled receptor (GPCR) superfamily, and contributes to DR. The current study is to uncover the roles of microRNA (miRNA) in thrombin-induced NF-kappa B activation and retinal endothelial functions.METHODS. Target prediction was performed using the TargetScan algorithm. Predicted target was experimentally validated by luciferase reporter assays. Human retinal endothelial cells (HRECs) were transfected with miRNA mimics or antimiRs and treated with thrombin. Expression levels of miR-146 and related protein-coding genes were analyzed by quantitative (q)RT-PCR. Functional changes of HRECs were analyzed by leukocyte adhesion assays.RESULTS. We identified that caspase-recruitment domain (CARD)-containing protein 10 (CARD10), an essential scaffold/adaptor protein of GPCR-mediated NF-kappa B activation pathway, is a direct target of miR-146. Thrombin treatment resulted in NF-kappa B-dependent upregulation of miR-146 in HRECs; while transfection of miR-146 mimics resulted in significant downregulation of CARD10 and prevented thrombin-induced NF-kappa B activation, suggest that a negative feedback regulation of miR-146 on thrombin-induced NF-kappa B through targeting CARD10. Furthermore, overexpression of miR-146 prevented thrombin-induced increased leukocyte adhesion to HRECs.CONCLUSIONS. We uncovered a novel negative feedback regulatory mechanism on thrombin-induced GPCR-mediated NF-kappa B activation by miR-146. In combination with the negative feedback regulation of miR-146 on the IL-1R/toll-like receptor (TLR)-mediated NF-kappa B activation in RECs that we reported previously, our results underscore a pivotal, negative regulatory role of miR-146 on multiple NF-kappa B activation pathways and related inflammatory processes in DR.