miR-146a抑制糖尿病视网膜色素上皮细胞损伤的机制

Inhibitory Effect of miR-146a on Diabetic Retinal Pigment Epithelial Cell Injury by Regulating Expression of Interleukin-1 Receptor-related Kinase-1

【目的】探讨miR-146a对人糖尿病视网膜色素上皮细胞(hRPE)损伤的影响及靶向调控机制。【方法】以高糖培养hRPE构建糖尿病hRPE损伤模型,hRPE中转染miR-146a mimics,用高糖培养液培养以后,Realtime PCR检测miR-146a表达变化。黄嘌呤氧化法检测SOD活性,硫代巴比妥酸法检测MDA含量。流式细胞术检测细胞凋亡。靶基因预测白介素1受体相关激酶1(IRAK1)可能是miR-146a的靶基因,构建荧光素酶报告系统鉴定靶向关系。以Western blot检测上调miR-146a对IRAK1表达影响。在hRPE中共转染pcDNA3.1-IRAK1和miR-146a mimics,用上述方法检测IRAK1对miR-146a调控高糖条件下hRPE中MDA、SOD和细胞凋亡的影响。【结果】高糖处理后的hRPE中miR-146a表达下调,细胞MDA含量升高,SOD活性降低,细胞凋亡率升高。miR-146a mimics转染后可以提高高糖条件下hRPE中miR-146a的表达水平,减少细胞中MDA含量,提高SOD活性,减少细胞凋亡。荧光素酶报告系统鉴定miR-146a靶向调控IRAK1,并且上调miR-146a抑制IRAK1蛋白表达。pcDNA3.1-IRAK1和miR-146a mimics共转染可以逆转上调miR-146a对高糖条件下hRPE中MDA、SOD和细胞凋亡的影响。【结论】miR-146a靶向下调IRAK1表达减轻糖尿病hRPE损伤。

【Objective】To investigate effects of miR-146a on human diabetic retinal pigment epithelial cell injury and novel mechanism.【Methods】Diabetic retinal pigment epithelial cell injury model was established by culturing human retinal pigment epithelial cells(hRPE)in high glucose medium.When hRPE cells was transfected with miR-146a mimics and cultured with high glucose medium,the expression of miR-146a was detected by real-time PCR.The activity of SOD was measured by xanthine oxidation.The content of MDA was detected by thiobarbituric acid.Apoptosis was detected by flow cytometry.Gene prediction showed that interleukin-1 receptor related kinase-1(IRAK1)was the possible target gene of miR-146a.So the luciferase reporter system was constructed to identify the targeting relationship.Western blot was further used to detect the effect of up-regulated miR-146a on IRAK1 expression.Finally,when both pcDNA3.1-IRAK1 and miR-146a mimics were co-transfected into hRPE cells,the effects of IRAK1 on MDA,SOD and cell apoptosis in hRPE cells regulated by microRNA-146 under high glucose condition were detected by the above methods.【Results】The expression of miR-146a was downregulated in hRPE cells after high glucose treatment.In the same condition,the content of MDA increased,SOD activity decreased and cell apoptosis rate increased in hRPE cells.Transfection of miR-146a mimics increased the expression level of miR-146a in hRPE cells under high glucose condition,therefore reduced cellular content of MDA,increased SOD activity and reversed cell apoptosis.The luciferase reporter system showed that miR-146a targeted IRAK1 gene,where the inhibitory IRAK1 protein expression was caused by miR-146a up-regulation.Co-transfection of pcDNA3.1-IRAK 1 and miR-146a mimics reversed the effects of miR-146a on MDA,SOD and cell apoptosis in retinal pigment epithelial cells under high glucose condition.【Conclusion】miR-146a down-regulation of IRAK1 expression attenuates the damage of diabetic retinal pigment epithelial cells.