miRNA-30C减轻高糖诱导H9c2大鼠心肌细胞损伤研究

miRNA-30c attenuates high glucose-induced cardiomyocyte injury in H9c2 rats

目的探究微小RNA-30c(miRNA-30c,miR-30c)是否能够通过调控p53来减轻高糖诱导的H9c2大鼠心肌细胞损伤。方法应用35 mM高糖(high glucose,HG)培养H9c2大鼠心肌细胞48 h,建立高糖诱导的心肌细胞损伤模型,5.0 mM葡萄糖处理48 h作为阴性对照(NG组),检测高糖环境对大鼠心肌细胞miR-30c表达水平的影响。然后将miR-30c mimics和阴性对照mimics control转染到H9c2心肌细胞内,转染后各组细胞给予35 mM HG孵育48 h。根据不同的处理方法将所有细胞分为四组:5.0 mM葡萄糖对照组(NG组);35 mM葡萄糖刺激组(HG组);转染miR-30c mimics再进行35 mM高糖刺激组(miR-30c mimics+HG组);转染mimics control再进行35 mM高糖刺激组(mimics control+HG组)。应用CCK-8检测各组心肌细胞的活力,DCFH-DA检测各组细胞活性氧水平的高低,JC-1荧光探针检测各组活细胞线粒体膜电位高低,Annexin V-FITC试剂盒检测各组凋亡细胞的情况。Western blot技术检测并分析各组心肌细胞中相关的凋亡蛋白p53、Bcl-2和Cleaved Caspase-3的表达水平,以β-actin为内参。结果高糖处理可以明显下调H9c2细胞中miR-30c表达,降低细胞活力和线粒体膜电位的表达水平,提高细胞内活性氧和凋亡的水平,促进凋亡相关蛋白p53和Cleaved Caspase-3的表达,抑制抗凋亡相关蛋白Bcl-2的表达;miR-30c模拟物转染后可以抑制上述高糖诱导的H9c2细胞损伤。结论在高糖诱导的H9c2大鼠心肌细胞中miR-30c低表达,而p53蛋白高表达;过表达的miR-30c可负性调节p53蛋白,可减轻高糖诱导的H9c2大鼠心肌细胞凋亡损伤。

Objective To investigate the effect of miR-30c on myocardial cell injury,and to verify whether miR-30c could negatively affect cardiomyocyte apoptosis by regulating p53 protein.Methods H9c2 rat cardiomyocytes were cultured with 35 mM high glucose(HG)for 48 hours to establish a high glucose induced cardiomyocyte injury model.Cells treated with 5.0 mM glucose for 48 hours were selected as a negative control(NG).The effect of high glucose environment on the expression level of miR-30c in H9c2 cardiomyocytes was detected.Then miR-30c mimics and negative control mimics were transfected into H9c2 cardiomyocytes,and the cells in each group were incubated with 35 mM HG for 48 hours after transfection.All cells were divided into four groups according to different treatment methods:5.0 mM glucose control(NG),35mM glucose stimulation group(HG),transfection of miR-30c mimics and 35mM high glucose stimulation group(miR-30c mimics+HG),transfection of mimics control and 35 mM high glucose stimulation group(mimics control+HG).CCK-8 was used to detect the viability of cardiomyocytes in each group,DCFH-DA was used to detect the level of reactive oxygen species in each group,JC-1 fluorescent probe was used to detect the mitochondrial membrane potential of living cells in each group and Annexin V-FITC kit was used to detect the apoptosis of cells in each group.Then,the expression levels of related apoptotic proteins p53,Bcl-2 and cleaved caspase-3 in cardiomyocytes of each group were detected and analyzed by Western blot.Results High glucose treatment significantly downregulated the expression of miR-30c in H9c2 rat cardiomyocytes,reduced the expression levels of cell viability and mitochondrial membrane potential,increased the levels of intracellular reactive oxygen species and apoptosis,promoted the expression of apoptosis-related proteins p53 and Cleaved Caspase-3,and inhibited the expression of anti-apoptosis-related protein Bcl-2.Transfection of miR-30c mimics inhibited the above-mentioned high glucose-induced H9c2 cell damage.Conclusion In H9c2 rat cardiomyocytes induced by high glucose,miR-30c was expressed at a low level,while p53 protein was expressed at a high level.Overexpression of miR-30c could negatively regulate p53 protein,which proves that it can reduce the cardiomyocyte apoptosis injury induced by high glucose in H9c2 rats.