Cowan Colleen, Muraleedharan Chithra K, O'Donnell James J, Singh Pawan K, Lum Hazel, Kumar Ashok, Xu Shunbin
Department of Pharmacology, Rush University Medical Center, Chicago, Illinois, United States.
Department of Ophthalmology, Kresge Eye Institute, School of Medicine, Wayne State University, Detroit, Michigan, United States Department of Anatomy & Cell Biology, School of Medicine, Wayne State University, Detroit, Michigan, United States.
Invest Ophthalmol Vis Sci. 2014 Jul 1;55(8):4944-51. doi: 10.1167/iovs.13-13631.
Nuclear factor-κB (NF-κB), a key regulator of immune and inflammatory responses, plays important roles in diabetes-induced microvascular complications including diabetic retinopathy (DR). Thrombin activates NF-κB 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-κB activation and retinal endothelial functions.
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.
We identified that caspase-recruitment domain (CARD)-containing protein 10 (CARD10), an essential scaffold/adaptor protein of GPCR-mediated NF-κB activation pathway, is a direct target of miR-146. Thrombin treatment resulted in NF-κB-dependent upregulation of miR-146 in HRECs; while transfection of miR-146 mimics resulted in significant downregulation of CARD10 and prevented thrombin-induced NF-κB activation, suggest that a negative feedback regulation of miR-146 on thrombin-induced NF-κB through targeting CARD10. Furthermore, overexpression of miR-146 prevented thrombin-induced increased leukocyte adhesion to HRECs.
We uncovered a novel negative feedback regulatory mechanism on thrombin-induced GPCR-mediated NF-κB activation by miR-146. In combination with the negative feedback regulation of miR-146 on the IL-1R/toll-like receptor (TLR)-mediated NF-κB activation in RECs that we reported previously, our results underscore a pivotal, negative regulatory role of miR-146 on multiple NF-κB activation pathways and related inflammatory processes in DR.
核因子-κB（NF-κB）是免疫和炎症反应的关键调节因子，在糖尿病诱导的微血管并发症（包括糖尿病视网膜病变，DR）中起重要作用。凝血酶通过蛋白酶激活受体（PAR）-1激活NF-κB，PAR-1是G蛋白偶联受体（GPCR）超家族的成员之一，并且与DR的发生有关。本研究旨在揭示微小RNA（miRNA）在凝血酶诱导的NF-κB激活及视网膜内皮细胞功能中的作用。
使用TargetScan算法进行靶标预测。通过荧光素酶报告基因检测对预测的靶标进行实验验证。用miRNA模拟物或抗miRNA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和相关蛋白质编码基因的表达水平。通过白细胞黏附试验分析HREC的功能变化。
我们发现含半胱天冬酶招募结构域（CARD）的蛋白10（CARD10）是GPCR介导的NF-κB激活途径的重要支架/衔接蛋白，是miR-146的直接靶标。凝血酶处理导致HREC中miR-146的NF-κB依赖性上调；而转染miR-146模拟物导致CARD10显著下调，并阻止了凝血酶诱导的NF-κB激活，这表明miR-146通过靶向CARD10对凝血酶诱导的NF-κB具有负反馈调节作用。此外，miR-146的过表达阻止了凝血酶诱导的白细胞与HREC黏附增加。
我们发现了miR-146对凝血酶诱导的GPCR介导的NF-κB激活的一种新的负反馈调节机制。结合我们之前报道的miR-146对视网膜内皮细胞中白细胞介素-1受体/ Toll样受体（TLR）介导的NF-κB激活的负反馈调节，我们的结果强调了miR-146在DR中对多种NF-κB激活途径及相关炎症过程的关键负调节作用。
