miRNA-let-7c在逆转大鼠糖尿病视网膜病变中的作用  

Effects of microrna-let-7c on reversing diabetic retinopathy in rats

作　　者：欧玉仑[1] 邝国平[1] 周小平[1] OU Yulun;KUANG Guoping;ZHOU Xiaoping(Department of Ophthalmology,Chenzhou NO.1 People’s Hospital,Chenzhou 423000,Hunan Province,China)

机构地区：[1]郴州市第一人民医院眼科,湖南省郴州市423000

出　　处：《眼科新进展》2020年第7期619-624,共6页Recent Advances in Ophthalmology

摘　　要：目的探讨MicroRNA-let-7c(miRNA-let-7c)在逆转糖尿病视网膜病变(diabetic retinopathy,DR)中的作用。方法制作DR大鼠模型48只,分为6组空白模型组、miRNA-let-7c模拟物组、Anti-miRNA-let-7c组、阴性对照(NC)组、Anti-miRNA-let-7c+Anti-STAT3组、Anti-STAT3组;同时选择8只正常大鼠作为正常组。采用实时荧光定量PCR检测各组大鼠视网膜和转染后的293T细胞中miRNA-let-7c和STAT3的mRNA表达水平,Western blot检测各组大鼠视网膜中STAT3、VEGFA、BAX及Bcl蛋白水平,通过HE染色计数大鼠视网膜血管内皮细胞的数目以及荧光素酶报告基因分析miRNA-let-7c与STAT3的直接作用。结果各组大鼠视网膜和转染后的293T细胞中miRNA let-7c与STAT3的mRNA表达呈负相关(r=-0.906,P<0.001)。与NC组的血管内皮细胞核(22±3)个相比,Anti-STAT3组的血管内皮细胞核(14±3)个明显减少(P<0.05);而Anti-miRNA-let-7c组血管内皮细胞核(58±6)个显著增加(P<0.05)。与NC组相比,miRNA-let-7c组和Anti-STAT3组大鼠视网膜中的STAT3、VEGFA和Bax蛋白表达显著降低(P<0.05);而Anti-miRNA-let-7c组大鼠视网膜中的STAT3、VEGFA和Bax蛋白表达显著升高(P<0.05)。此外,miRNA-let-7c组miRNA-let-7c mRNA表达为2.24±0.25,较NC组miRNA-let-7c mRNA表达(1.32±0.17)显著升高(P<0.05),而Anti-miRNA-let-7c组miRNA-let-7c mRNA表达(0.62±0.05)显著降低(P<0.05)。双荧光素酶报告基因检测结果显示,STAT33’UTR-WT的相对荧光素酶活性为0.55±0.03,低于WT+NC组的1.04±0.18,差异有统计学意义(P<0.05),表明STAT3是miRNA-let-7c的靶基因。结论miRNA-let-7c的过表达可能下调STAT3的表达,从而阻止大鼠DR模型的发展。Objective To investigate the mechanism of miRNA-let-7c in reversing diabetic retinopathy(DR)of rats.Methods A total of 48 DR rat models were established and they were divided into 6 groups(with 8 rats in each group)blank model group,the miRNA-Let-7c group,the anti-miRNA-Let-7c group,the negative control(NC)group,the Anti-STAT3 group,and the anti-miRNA-Let-7c+Anti-STAT3 group respectively,while additional 8 normal rats were selected as the normal group.The qRT-PCR was used to detect the expression of miRNA-Let-7c and STAT3 mRNA.The STAT3,VEGFA,Bax and Bcl protein expression was measured using Western blot.The number of retinal vascular endothelial cells was counted by HE staining,and the direct function of miRNA-let-7c and STAT3 was analyzed by luciferase reporter gene.Results In all groups,MiRNA Let-7c was negatively correlated with STAT3 mRNA expression in rat retina and transfected 293T cells(r=-0.906,P<0.001).Compared with the nucleus of vascular endothelial cells in the NC group(22±3)cells,the nucleus of vascular endothelial cells in the Anti-STAT3 group was(14±3)cells,which was significantly reduced(P<0.05);the Anti-miRNA-let-7c group was(58±6)cells,and the vascular endothelial cell nuclei increased significantly(P<0.05).Compared with NC group,the expression of STAT3,VEGFA and Bax protein in miRNA-let-7c group and Anti-STAT3 group decreased significantly(all P<0.05),while the expression of STAT3,VEGFA and Bax protein in Anti-miRNA-let-7c group increased significantly(all P<0.05).In addition,miRNA-let-7c mRNA expression in the miRNA-let-7c group was(2.24±0.25),which was significantly higher than that in the NC group(1.32±0.17)(P<0.05),while the Anti-miRNA-let-7c group(0.62±0.05)was significantly reduced(P<0.05).The detection result of the double luciferase reporter gene STAT33’UTR-WT had a relative luciferase activity(0.55±0.03)lower than that of the WT+NC group(1.04±0.18),and the difference was statistically significant(P<0.05),indicating STAT3 is a target gene of miRNA-let-7c.Conclusion Overexpres

关 键 词：MicroRNA-let-7c STAT3 糖尿病视网膜病变 内皮细胞核 

分 类 号：R774[医药卫生—眼科]