芍药醇调控miR-122-5p/GSTM1分子轴对缺血再灌注心肌细胞存活的影响

Effect of Paeonol on the Survival of Ischemia and Reperfusion Induced Cardiomyocytes by Regulating miR-122-5p/GSTM1 Molecular Axis

目的:探讨芍药醇对缺血再灌注心肌细胞存活的影响,并探讨其机制是否与调控miR-122-5p/谷胱甘肽转移酶M1(GSTM1)分子轴有关。方法:将H9C2细胞分为正常对照(NC)组、缺血/再灌注(I/R)组、I/R+低-中-高芍药醇组、I/R+anti-miRcon组、I/R+anti-miR-122-5p组、I/R+芍药醇+miR-con组、I/R+芍药醇+miR-122-5p组、I/R+芍药醇+miR-122-5p+pc DNA-con组、I/R+芍药醇+miR-122-5p+pc DNA-GSTM1组,采用四甲基偶氮唑蓝(MTT)法检测细胞活力;实时荧光定量PCR(RT-qPCR)和蛋白质印记(Western blot)检测miR-122-5p、GSTM1表达;双荧光素酶报告基因实验和Western blot确定miR-122-5p对GSTM1的靶向作用。结果:I/R处理后,H9C2细胞存活率、GSTM1表达降低,miR-122-5p表达升高(P<0.05)。芍药醇干预后,H9C2细胞存活率、GSTM1表达升高,miR-122-5p表达降低(P<0.05)。抑制miR-122-5p表达后H9C2细胞存活率升高,从而减轻I/R对H9C2细胞的抑制作用(P<0.05)。过表达miR-122-5p可逆转芍药醇对I/R处理下H9C2细胞存活的保护作用(P<0.05)。过表达GSTM1可逆转miR-122-5p对芍药醇干预的I/R处理下H9C2细胞存活的影响(P<0.05)。结论:GSTM1是miR-122-5p的靶基因,miR-122-5p对GSTM1具有负性调控作用。芍药醇可提高I/R条件下心肌细胞存活率,其机制与下调miR-122-5p/GSTM1分子轴有关。

Objective: To investigate the effect of paeonol on the survival of cardiomyocytes induced by ischemia and reperfusion,and to investigate whether its mechanism is related to the regulation of the molecular axis of miR-122-5p/glutathione transferase M1(GSTM1). Methods: H9C2 cells were divided into normal control(NC) group,ischemia/reperfusion(I/R) group,I/R + low,medium and high dose paeonol groups,I/R + anti-miR-con group,I/R + anti-miR-122-5p group,I/R + paeonol + miR-con group,I/R + paeonol + miR-122-5p group,I/R + paeonol + miR-122-5p + pc DNA-con group and I/R + paeonol + miR-122-5p + pc DNAGSTM1 group. Methyl thiazolyl tetrazolium(MTT) assay was selected to detect the cell viability;real-time quantitative PCR(RTq PCR) and Western blot were utilized to detect miR-122-5p and GSTM1 expressions;dual luciferase reporter gene experiment and Western blot were performed to determine the targeting effect of miR-122-5p on GSTM1. Results: After I/R treatment,the survival rate of H9C2 cells and the expression of GSTM1 decreased,while the expression of miR-122-5p increased(P<0.05). After paeonol treatment,the survival rate of H9C2 cells and the expression of GSTM1 increased,while the expression of miR-122-5p decreased(P <0.05). After inhibiting the expression of miR-122-5p,the survival rate of H9C2 cells increased,thereby reducing the inhibitory effect of I/R on H9C2 cells(P<0.05). Overexpression of miR-122-5p reversed the protective effect of paeonol on the survival of H9C2 cells under I/R treatment(P<0.05). Overexpression of GSTM1 reversed the effect of miR-122-5p on the survival of H9C2 cells under I/R treatment with paeonol(P < 0.05). Conclusion: GSTM1 was a target gene of miR-122-5p,and miR-122-5p negatively regulated GSTM1 expression. Paeonol can increase the survival rate of cardiomyocytes under I/R conditions,and its mechanism is related to the down-regulation of miR-122-5p/GSTM1 molecular axis.