CAJ | 학술논문

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.
목적 연구미소RNA(miRNA)-195재당뇨병심기병심기비대중적작용급기궤제.방법 응용TargetScan5.1연건예측Smad7위miRNA-195발휘작용적잠재파점.분리원대SD유서심기세포진행배양,첩벽후장원대심기세포분위3조,정상대조조심기세포용농도위5 mmol/L포도당배양,고당대조조심기세포용농도위25 mmol/L포도당배양,실험조심기세포경miRNA-195억제제전염후용농도위25 mmol/L포도당배양.배양24、48화72 h시,우도치상차현미경하관찰심기세포형태학변화,병박조계산심기세포표면적,채용역전록실시정량취합매련반응(RT-PCR)검측심기세포miRNA-195화심기비대기인β-기구단백중련(β-MHC) mRNA적표체,채용단백질인적(Western blot)법검측심기세포중Smad7단백적표체,채용매련면역흡부시험(ELISA)검측각조세포배양상청액중전화생장인자(TGF)-β1농도.통과억제miRNA-195표체,관찰기대Smad7、β-MHC표체급심기비대적영향.결과 고당대조조각시점miRNA-195표체수평균고우정상대조조(P균＜0.05),배양48 h시고당대조조Smad7단백표체수평저우정상대조조(P＜0.05),이실험조miRNA-195표체수평저우고당대조조,Smad7단백표체수평칙고우고당대조조(P＜0.05).고당대조조심기세포경고당처리후,Smad7표체여miRNA-195표체정현저부상관(상관계수위-0.945,P＜0.05).배양48 h시고당대조조TGF-β1농도고우정상대조조(P＜0.05),여상동시간점고당대조조비교,실험조TGF-β1농도칙균교저(P균＜0.05).수배양시간적연장,고당자격사심기세포표면적、β-MHC mRNA표체축점증고,기변화추세여miRNA195표체정황일치.결론 miRNA-195재당뇨병심기병심기비대적발생、발전과정중구유중요작용,기궤제가능여하조파단백Smad7표체유관.
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.