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Shunbin Xu, Colleen Cowan, James O'Donnell, Hazel Lum; microRNA-146 Inhibits Thrombin-induced NF-κB Activation and Subsequent Inflammatory Responses in Human Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1040.
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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 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 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 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 down-regulation of CARD10 and prevented thrombin-induced NF-κB activation, suggest 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/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, supporting that miR-146 is a potential therapeutic target for DR and other diabetic microvascular complications.
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