miR21对心脏微血管内皮细胞PDCD4/AP1通路的调节作用  

The Regulation of miR-21 on the PDCD4/AP1 Pathway in Cardiac Microvascular Endothelial Cells

作　　者：虞勇[1] 虞莹[1] 刘桂剑 王兴岗 郭棋[1] 邹云增[1] 陈瑞珍[1] 

机构地区：[1]复旦大学附属中山医院上海市心血管病研究所卫生部病毒性心脏病重点实验室,上海200032 [2]苏州大学附属第一医院心内科,苏州市215006

出　　处：《中国分子心脏病学杂志》2015年第6期1534-1538,共5页Molecular Cardiology of China

基　　金：国家自然科学基金资助课题(31070786)

摘　　要：目的探讨柯萨奇B3病毒(CVB3)感染离体培养的大鼠心脏微血管内皮细胞(CMVECs)后,miR-21对PDCD4和转录因子AP1构成的调节通路的影响。方法 (1)CVB3感染CMVECs 48h,实时荧光定量PCR检测各组细胞中miR-21表达;(2)CVB3病毒感染CMVECs48h后,Western Blot法检测各组细胞中PDCD4蛋白表达;(3)CMVECs瞬时转染miR-21模拟物48h后,Western Blot法检测各组细胞中PDCD4蛋白表达;(4)双荧光素酶报告基因法研究miR-21与PDCD4基因靶向结合位点;(5)CM V ECs细胞共转染PDCD4和AP1-Luc质粒,检测AP1-Luc荧光素酶活性以观察PDCD4基因对AP1活性的影响;(6)提取各组细胞核蛋白,电泳迁移率改变实验(EMSA实验)检测AP1和DNA的结合活性。结果 (1)CMVECs感染CVB3病毒48小时后,与对照组比较,病毒感染组miR-21表达上调4.12倍;(2)CMVECs感染CVB3病毒48小时后,与对照组比较,病毒感染组PDCD4蛋白表达明显下调;(3)CM V ECs过表达miR-21后PDCD4蛋白表达下调;(4)CM V ECs过表达miR-21后显著抑制野生型PDCD4基因3’UTR活性,而对突变型3’UTR活性无影响;(5)CMVECs过表达PDCD4基因48h后AP1-Luc荧光素酶活性与对照组相比明显下调;CMVECs过表达miR-21 48h后AP1-Luc荧光素酶活性与对照组相比显著上调;(6)EMSA实验结果显示CMVECs过表达PDCD4 48h后AP1与DNA结合活性明显下调,过表达miR-21后AP1与DNA结合活性明显上调。结论 CVB3感染CMVECs后上调miRNA-21表达。miR-21通过与靶基因PDCD4的3’非翻译区(3’UTR)靶向结合抑制PDCD4表达、上调A P1活性及A P1与DNA结合活性。Objective In this study we explored the role of miR-21 on the PDCD4/AP1 pathway in Cardiac Microvascular endothelial Cells（ CMVECs） after Coxsackie virus B3（CVB3） infection in vitro. Methods（1） After 48 hours of virus infection,the expression of miR-21 was detected in each group by RT-PCR;（2） After 48 hours of virus infection,expression of PDCD4 protein was detected in the cells of each group by Western Blot;（3） miR-21 mimics was transfected into CMVECs for 48 hours by liposome transfection, expression of PDCD4 protein was detected in the cells of each group by Western Blot;（4） To study the targeting binding site of miR-21 and PDCD4 gene by using double f luorescent enzyme reporter gene method;（5） The AP1-Luc plasmid and PDCD4 gene were cotransfected into CMVECs and the luciferase activity was detected;（6） The DNA binding activities of AP1 in the cells were measured by the electrophoretic mobility shift assay（ EMSA）. Results（1） Compared with control, the expression of miR-21 was 4.12 times increased in CVB3 infection group;（2） Compared with control, the expression of PDCD4 protein was markedly decreased in CVB3 infection group;（3）Overexpression of miR-21 leads to expression of PDCD4 protein was markedly decreased;（4） Overexpression of miR-21 leads to luciferase activity of 3＇UTR of wild type gene was markedly decreased, which was not signif icantly different in 3＇ mut UTR group;（5） Overexpression of PDCD4 gene in CMVECs for 48 h, compared with control, AP1-luciferase activity markedly decreased; Overexpression of miR-21 in CMVECs for 48 h,compared with control, AP1-luciferase activity markedly increased;（6） EMSA showed that stimulation of CMVECs by miR-21 markedly increased DNA binding activity. Conclusion CVB3 inhibited PDCD4 gene expression in miR-21 dependent manner, PDCD4 gene down-regulates the transcription activity of AP1 in CMVECs.

关 键 词：MIR-21 柯萨奇B3病毒 心脏微血管内皮细胞 PDCD4 AP1 

分 类 号：R542.21[医药卫生—心血管疾病]