微小核糖核酸miR-766通过靶向调控环腺苷酸活化交换蛋白1促进高糖培养下心肌细胞凋亡  

MicroRNA-766 contributes to high glucose-induced cardiomyocyte apoptosis by targeting exchange protein directly activated by cyclic adenylic acid isoform 1

作　　者：孙振华 梁巍巍[1] 管洪宇[1] 李海[1] 何筱莹[1] 刘娟[1] 刘烈华[1] 李延兵[1] Sun Zhenhua;Liang Weiwei;Guan Hongyu;Li Hai;He Xiaoying;Liu Juan;Liu Liehua;Li Yanbing(Department of Endocrinology and Diabetes Center,The First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080,China)

机构地区：[1]中山大学附属第一医院内分泌科,广州510080

出　　处：《中华糖尿病杂志》2018年第8期515-520,共6页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：中山大学临床医学研究5010计划项目

摘　　要：目的探讨微小核糖核酸miR-766在不同葡萄糖浓度培养的心肌细胞中的表达水平，明确其对心肌细胞凋亡的影响及相关分子机制。方法在不同葡萄糖浓度条件下（正常糖5mmol／L、高糖15、25及35mmol／L）培养的心肌细胞中，采用实时荧光定量聚合酶链式反应（PCR）检测miR-766的表达差异，以流式细胞术检测各组心肌细胞凋亡情况，Western blot法检测各组环腺苷酸活化交换蛋白l（Epac-1）、Bax蛋白、半胱氨酸天冬氨酸特异性蛋白酶-3（caspase-3）活化片段蛋白表达水平。采用生物信息学预测miR-766的靶基因，并进一步采用双荧光素酶报告基因法进行验证。组间数据比较采用t检验。结果与5mmol／L相比，miR-766在15、25及35mmol／L组的心肌细胞中表达上调（3．40±0．38，4．61±0．34，6．17±0．42比0．97±0．04，t=11．13l-21．493，P〈0．05），而转染miR-766抑制剂后，细胞的凋亡率明显降低[（16．43±0．31）％比（26．45±0-31）％，t=39．566，P〈0．05]；Western blot结果表明，在人心肌细胞中miR-766高表达者Epac-1的蛋白表达减少（0．48±0．07比1．00±O．12，t=6．695，P〈0．05），而抑制miR-766的表达后Epac-1的表达增加（1．23±0．12比0．70±0．10，t=5．884，P〈0．05）；荧光素酶报告基因结果表明，miR-766模拟物或抑制剂与Epae-1基因3＇-非翻译区野生型报告基因共转染时，报告基因荧光素酶活性明显变化（3．22±0．07比5．01±0．96，6．98±0．61比5．01±O．96，t=-3．239～-3．008，P〈0．05）；在高糖培养条件下抑制miR-766表达能够部分拮抗高糖对Epac-1蛋白表达的抑制作用（0．80±0．05比0．53±0．05，t=6．810，P〈0．05）；在miR-766模拟物处理的H9e2细胞中回补Epae-1，easpase-3活化片段蛋白的表达则明显降低（O．67±0．07比1．07±0．12，t=-5．050，P〈0．05）。结论在高糖培养的心肌细胞中高表达的miR-7Objective To investigate the expression levels and functional role of microRNA 766 （miR-766） in cardiomyocytes treated with high glucose conditions. Method Cardiomyocytes were cultured with different concentrations of glucose levels at 5.0 mmol/L （Controls）, 15, 25 or 35 retool/L, respectively. The expression of miR-766 was detected by real-time PCR, apoptosis was detected by flow cytometry, and the expressions of exchange protein directly activated by cyclic adenylic acid isoform 1 （Epac-1）, Bax and cleaved cysteinyl aspartate specific proteinase-3 （caspase-3） were determined by Western blot. The target gene of miR-766 calculated by bioinformatics was further determined by using dual luciferase system. T test was used for comparison between groups. Results Compared with the controls, the expressions of miR-766 were significantly upregulated in different high glucose （HG）-stimulated eardiomyocytes （3.40 ± 0.38, 4.61±0.34, 6.17 ±0.42 vs 0.97±0.04, t=11.131-21.493, all P〈0.05）. Inhibition of miR-766 in cardiomyocytes treated with HG resulted in reduced apoptosis [（16.43 ± 0.31）% vs （26.45 ± 0.31）% ,t=-39.566, P〈0.05]. Overexpression of miR-766 decreased the protein levels of Epac-1 （0.48±0.07 vs 1.00± 0.12, t=-6.695, P〈0.05）, and inhibition of miR-766 increased the protein levels of Epac-1 （1.23±0.12 vs 0.70±0.10, t=5.884, P〈0.05）. Luciferase reporter assays showed that overexpression of miR-766 significantly decreased the luciferase activity of wild-type reporter, and inhibition of miR-766 significantly increased the luciferase activity of wild-type reporter （3.22 ± 0.07 vs 5.01 ± 0.96; 6.98 ± 0.61 vs 5.01 ± 0.96, t=-3.239- -3.008, P〈0.05）. Moreover, transfection of miR-766 inhibitor could partly reverse the suppression of Epac-1 by HG in H9c2 cells （0.80±0.05 vs 0.53±0.05, t=6.810, P〈0.05）. Restoration of Epac-1 in miR-766-treated H9c2 cells reversed the effects of miR-766 on cleaved caspase-3 expression （0.67 ± 0.07 vs 1.07 ± 0.12, t

关 键 词：糖尿病心肌病 细胞凋亡 微RNAS miR-766 环腺苷酸活化交换蛋白1 

分 类 号：R542.2[医药卫生—心血管疾病]