摘要
目的 探讨微小核RNA-29a(miR-29a)在缺血再灌注引起的心肌细胞损伤中的功能和分子机制。方法 体外培养H9c2大鼠心肌细胞,并将其分为正常对照组(正常培养的H9c2细胞)、缺氧再灌注(H/R)组(缺氧处理4 h,继而复氧4 h)、H/R+miR-29a inhibitor组(在H/R组基础上转染miR-29a抑制剂)、H/R+miR-29a inhibitor+LY294002组(在H/R+miR-29a inhibitor组基础上加用2μg·mL-1抑制剂LY294002)。通过实时荧光定量聚合酶链反应检测miR-29a和磷脂酰肌醇-3-羟激酶(PI3K)的表达水平;使用细胞计数试剂盒8(CCK-8)试剂盒检测细胞活力;通过流式细胞术和原位末端标记(TUNEL)实验检测细胞凋亡;使用试剂盒检测乳酸盐脱氢酶(LDH)和肌酸激酶(CK)活性;通过双荧光素酶报告实验验证miR-29a和PI3K的靶向关系。结果 对照组、H/R组和H/R+miR-29a inhibitor组细胞miR-29a表达水平分别为1.05±0.02、4.13±0.15、1.56±0.21,细胞活力分别为(100.00±0.08)%、(50.02±0.13)%和(83.65±0.04)%,CK含量分别为(71.46±0.08)、(140.22±0.14)和(98.86±0.25)U·L-1,NC组上述指标和H/R+miR-29a inhibitor组与H/R组比较,差异均有统计学意义(P<0.05,P<0.01)。H/R+miR-29a inhibitor组中磷酸化p-PI3K蛋白(0.73±0.09)和磷酸化蛋白激酶B (p-AKT)蛋白表达(0.82±0.04)显著高于H/R组中p-PI3K蛋白(0.29±0.09)和p-AKT蛋白表达(0.39±0.24),差异均有统计学意义(均P<0.01),在加入PI3K/AKT信号通路抑制剂LY294002后p-PI3K和p-AKT的表达水平下调。结论 miR-29a可促进心肌细胞凋亡,敲降miR-29a可以保护心肌细胞免受H/R损伤,miR-29a对H/R心肌细胞的保护作用是通过激活PI3K/AKT通路实现的。
Objective To investigate the function and molecular mechanism of microRNA-29a(miR-29a)in myocardial cell injury induced by ischemia-reperfusion.Methods H9c2 rat cardiomyocytes were cultured in vitro and divided into normal control group(H9c2 cells in normal culture),hypoxia-reperfusion(H/R)group(hypoxia treatment for 4 h,followed by reoxygenation for 4 h),H/R+miR-29a inhibitor group(transfection of miR-29a inhibitor on the basis of H/R group),H/R+miR-29a inhibitor+LY294002 group(addition of 2μg·mL^(-1) inhibitor LY294002 on the basis of H/R+miR-29 inhibitor group).The expression levels of miR-29a and phosphatidylinositol 3-kinase(PI3K)were detected by real-time fluorescence quantitative polymerase chain reaction;cell counting kit-8(CCK-8)was used to detect cell viability;apoptosis was detected by flow cytometry and in situ end labeling(TUNEL)assay;the activities of lactate dehydrogenase(LDH)and creatine kinase(CK)were detected by kits;the targeting relationship between miR-29a and PI3K was verified by dual luciferase reporter assay.Results The expression of miR-29a in control group,H/R group,H/R+miR-29a inhibitor group and cells were 1.05±0.02,4.13±0.15,1.56±0.21,respectively;the cell viability were(100.00±0.08)%,(50.02±0.13)%and(83.65±0.04)%,respectively,and the CK content was(71.46±0.08),(140.22±0.14)and(98.86±0.25)U·L^(-1),respectively.There were significant differences in the above indexes between NC group and H/R+miR-29a inhibitor group and H/R group(P<0.05,P<0.01).The expression of phosphorylated PI3K(p-PI3K)protein(0.73±0.09)and phosphorylated protein kinase B(p-AKT)protein(0.82±0.04)in the H/R+miR-29a inhibitor group was significantly higher than that in the H/R group p-PI3K protein(0.29±0.09)and p-AKT protein expression(0.39±0.24),the differences were statistically significant(all P<0.01).After adding PI3K/AKT signaling pathway inhibitor LY294002,the expression of p-PI3K and p-AKT was down-regulated.ConclusionnmiR-29a can promote apoptosis of cardiomyocytes,and knocking down miR-29a can p
作者
孙小林
孟凡棣
贾政
耿玥
韦杰
SUN Xiao-lin;MENG Fan-di;JIA Zheng;GENG Yue;WEI Jie(Cardiac Great Vascular Surgery Intensive Care Unit,Yan'an Hospital of Kunming City,Kunming 650051,Yunnan Province,China)
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2023年第17期2477-2481,共5页
The Chinese Journal of Clinical Pharmacology
基金
云南省科技计划基金资助项目(202201AT070277)。
