微小RNA-195在糖尿病心肌病乳鼠心肌肥大中的作用及其机制  

Inhibition of microRNA195 attenuates high-glucose induced neonatal cardiomyocytes hypertrophy in vitro

作　　者：孔彪[1] 沈冬丽 芮涛[1] 张国辉[1] 

机构地区：[1]江苏大学附属人民医院心内科,镇江212002

出　　处：《中华心血管病杂志》2015年第8期712-717,共6页Chinese Journal of Cardiology

基　　金：国家自然科学基金（81370333）

摘　　要：目的 研究微小RNA（miRNA）-195在糖尿病心肌病心肌肥大中的作用及其机制.方法 应用TargetScan5.1软件预测Smad7为miRNA-195发挥作用的潜在靶点.分离原代SD乳鼠心肌细胞进行培养,贴壁后将原代心肌细胞分为3组,正常对照组心肌细胞用浓度为5 mmol/L葡萄糖培养,高糖对照组心肌细胞用浓度为25 mmol/L葡萄糖培养,实验组心肌细胞经miRNA-195抑制剂转染后用浓度为25 mmol/L葡萄糖培养.培养24、48和72 h时,于倒置相差显微镜下观察心肌细胞形态学变化,并拍照计算心肌细胞表面积,采用逆转录实时定量聚合酶链反应（RT-PCR）检测心肌细胞miRNA-195和心肌肥大基因β-肌球蛋白重链（β-MHC） mRNA的表达,采用蛋白质印迹（Western blot）法检测心肌细胞中Smad7蛋白的表达,采用酶联免疫吸附试验（ELISA）检测各组细胞培养上清液中转化生长因子（TGF）-β1浓度.通过抑制miRNA-195表达,观察其对Smad7、β-MHC表达及心肌肥大的影响.结果 高糖对照组各时点miRNA-195表达水平均高于正常对照组（P均＜0.05）,培养48 h时高糖对照组Smad7蛋白表达水平低于正常对照组（P＜0.05）,而实验组miRNA-195表达水平低于高糖对照组,Smad7蛋白表达水平则高于高糖对照组（P＜0.05）.高糖对照组心肌细胞经高糖处理后,Smad7表达与miRNA-195表达呈显著负相关（相关系数为-0.945,P＜0.05）.培养48 h时高糖对照组TGF-β1浓度高于正常对照组（P＜0.05）,与相同时间点高糖对照组比较,实验组TGF-β1浓度则均较低（P均＜0.05）.随培养时间的延长,高糖刺激使心肌细胞表面积、β-MHC mRNA表达逐渐增高,其变化趋势与miRNA195表达情况一致.结论 miRNA-195在糖尿病心肌病心肌肥大的发生、发展过程中具有重要作用,其机制可能与下调靶蛋白Smad7表达有关.Objective To investigate the effects of micro （mi）RNA-195 on high-glucose induced neonatal cardiomyocyte hypertrophy and to explore the related mechanism.Methods The potential target gene of miRNA-195 （Smad7） was predicted by TargetScanS.1 software.Cardiomyocytes were isolated from neonatal SD rats and cells were then randomly divided into three groups：cells treated by culture medium containing 5 mmol/L glucose （control group）,by culture medium containing 25 mmol/L glucose （high glucose group） and treated by culture medium containing 25 mmol/L glucose and miRNA-195 inhibitor transfection （miRNA-195 inhibitor group）.After 24,48,or 72 h of in vitro culture,the morphology of cardiomyocytes was examined under phase contrast microscope.Micrographs were captured and the cell surface was calculated.The mRNA expressions of miRNA-195 and myosin heavy chain β （β-MHC）,a biomarker for cardiomyocyte hypertrophy,in cardiomyocytes were detected by RT-PCR.The protein expression of Smad7 was determined by Western blot.The concentration of transforming growth factor-β1 （TGF-β1） in the supernatant of culture medium was measured by ELISA.Results Cross-sectional area of cardiomyocytes,expression of miRNA-195 and β-MHC and secretion of TGF-β1 were significantly increased in high glucose-treated cells （P 〈 0.05 vs.normal control）.The protein expression of Smad7 was significantly downregulated in cells exposed to high glucose for 48 h （P 〈 0.05 vs.normal control）.Downregulation of miRNA-195 partly reversed the high glucose-induced effects.The expression of Smad7 was negatively correlated with miRNA-195 in high glucose control group （correlation coefficient：-0.945,P 〈 0.05）.Conclusion Our results demonstrate that Smad7 could be the target gene of miRNA-195.miRNA-195 might play a crucial role in the development and progression of diabetic cardiomyopathy possibly through downregulating the expression of Smad7 and modulating TGF-β/Smad pathways.

关 键 词：糖尿病 心肌疾病 微RNAS 

分 类 号：R587.2[医药卫生—内分泌]