miR-146a调控人脐静脉内皮细胞的衰老及机制  

miR-146a regulates human umbilical vein endothelial cells senescence and its mechanism

作　　者：安雪青 吕健东[1] 苏锋[1] AN Xueqing;LV Jiandong;SU Feng(Tianjin Fifth Hospital,Tianjin 300450,China)

机构地区：[1]天津市第五中心医院

出　　处：《中国动脉硬化杂志》2018年第11期1127-1132,共6页Chinese Journal of Arteriosclerosis

基　　金：天津市卫生局科技基金项目(2013KZ020);滨海新区卫生局科技项目(2012BWKY006)

摘　　要：目的 探究miR-146a在人脐静脉内皮细胞衰老中的调节作用,并初步探究其机制。方法 以人脐静脉内皮ECV-304细胞为研究对象,连续传代建立ECV-304细胞复制性衰老模型,衰老相关β-半乳糖苷酶(SA-β-gal)法检测不同代次(P2、P6、P9、P12、P15代)细胞中衰老细胞比例,实时定量PCR法检测不同代次(P2、P6、P9、P12、P15代)细胞中miR-146a表达。靶基因预测软件预测miR-146a靶基因。细胞转染建立miR-146a表达上调的P12代细胞(Pre-miR146a组)和miR-146a表达下调的P12代细胞(Anti-miR146a组),并建立相应对照组,SA-β-gal法检测衰老细胞比例,Western blot检测NADPH氧化酶4(NOX4)蛋白表达。结果 P6、P9、P12和P15代细胞的衰老细胞比例明显高于P2代细胞(P<0.05),miR-146a表达则明显低于P2代细胞(P<0.05)。靶基因预测miR-146a的靶基因为NOX4;转染后,Pre-miR146a组衰老细胞比例明显低于对照组(P<0.05),Anti-miR146a组衰老细胞比例明显高于对照组(P<0.05);Pre-miR146a组NOX4蛋白表达明显低于对照组(P<0.05),Anti-miR146a组NOX4蛋白表达明显高于对照组(P<0.05)。结论 miR-146a在衰老的人脐静脉内皮细胞ECV-304中表达下降,上调miR-146a表达能够抑制ECV-304衰老,其机制与调节NOX表达有关。Aim To investigate the regulatory role of miR-146a in the senescence of human umbilical vein endothelial cells and its mechanism.Methods The replication aging model of human umbilical vein endothelial ECV-304 cells was established.Senescence-associated β-galactosidase (SA-β-gal) method was used to detect the ratio of senescent cells in different generations cells (P2, P6, P9, P12, P15 generation); real time quantitative PCR method was used to detect the level of miR-146a in different generations cells (P2, P6, P9, P12, P15 generation); Target gene prediction software was used to predict the target gene of miR-146a.The cells in P12 generation were transfected with Pre-miR146a, Pre-miR146a control, Anti-miR146a , and Anti-miR146a control; SA-β-gal method was used to detect the ratio of senescent cells; Western blot was used to detect the protein expression of NADPH oxidase 4(NOX4).Results The ratio of senescent cells in P6, P9, P12 and P15 generations was significantly higher than cells in P2 generation (P<0.05).And the expression of miR-146a in P6, P9, P12 and P15 generations was significantly lower than cells in P2 generation (P<0.05).Predicting the target gene of miR-146a was NOX4.After transfection, the ratio of senescent cells in Pre-miR146a group was significantly lower than control group (P<0.05), but it was significantly higher in Anti-miR146a group (P<0.05).The expression of NOX4 protein in Pre-miR146a group was significantly lower than control group (P<0.05), but it was significantly higher in Anti-miR146a group than control group (P<0.05).Conclusion The expression of miR-146a decreased in the aging human umbilical vein endothelial ECV-304 cells, the up-regulation of miR-146a expression can inhibit ECV-304 senescence, and its mechanism is related to the regulation of NOX expression.

关 键 词：MIR-146A 衰老 脐静脉内皮细胞 NADPH氧化酶4 

分 类 号：R543[医药卫生—心血管疾病]