LncRNA RMST靶向miR-27a-3p影响过氧化氢诱导心肌细胞损伤

Study on the Mechanism of LncRNA RMST Affects the Mechanism of Hydrogen Peroxide-Induced Cardiomyocyte Damage by Targeting miR-27a-3p

为了探讨LncRNA RMST对过氧化氢(H_(2)O_(2))诱导的心肌细胞损伤的影响及其可能的作用机制,该研究采用H_(2)O_(2)诱导大鼠心肌细胞H9C2建立细胞氧化损伤模型,随机分组:si-NC+200μmol/L H_(2)O_(2)组(即培养基中加入200μmol/L H_(2)O_(2)干预2 h)、si-LncRNA RMST+200μmol/L H_(2)O_(2)组、miR-NC+200μmol/L H_(2)O_(2)组、miR-27a-3p+200μmol/L H_(2)O_(2)组、anti-miR-NC+si-LncRNA RMST+200μmol/L H_(2)O_(2)组、anti-miR-27a-3p+si-LncRNA RMST+200μmol/L H_(2)O_(2)组。qRT-PCR法检测LncRNA RMST和miR-27a-3p的表达量;利用相应试剂盒检测细胞培养液中LDH的水平,以及细胞内MDA、SOD的水平;流式细胞术检测细胞凋亡率;双荧光素酶报告实验检测LncRNA RMST和miR-27a-3p的靶向关系;Western blot法检测Cleaved-caspase-3、Bax蛋白表达量。结果显示,与si-NC+200μmol/L H_(2)O_(2)组比较,si-LncRNA RMST+200μmol/L H_(2)O_(2)组中的MDA、LDH的水平、细胞凋亡率以及Cleaved-caspase-3、Bax蛋白水平降低(P<0.05),SOD的水平升高(P<0.05);LncRNA RMST可靶向调节miR-27a-3p的表达;与miR-NC+200μmol/L H_(2)O_(2)组比较,miR-27a-3p+200μmol/L H_(2)O_(2)组中的MDA、LDH的水平、细胞凋亡率以及Cleaved-caspase-3、Bax蛋白水平降低(P<0.05),SOD的水平升高(P<0.05);与anti-miR-NC+si-LncRNA RMST+200μmol/L H_(2)O_(2)组比较,anti-miR-27a-3p+si-LncRNA RMST+200μmol/L H_(2)O_(2)组MDA、LDH的水平、细胞凋亡率以及Cleaved-caspase-3、Bax蛋白水平升高(P<0.05),SOD的水平降低(P<0.05)。这提示,干扰LncRNA RMST表达可通过靶向miR-27a-3p抑制心肌细胞氧化应激反应及凋亡进而减轻H_(2)O_(2)诱导的心肌细胞损伤。

To explore the effect of LncRNA RMST on the damage of cardiomyocytes induced by hydrogen peroxide(H_(2)O_(2))and its possible mechanism,H_(2)O_(2)was used to induce rat cardiomyocyte H9C2 to establish a cell oxidative damage model.si-NC,si-LncRNA RMST,miR-NC,miR-27a-3p mimics,anti-miR-NC and si-LncRNA RMST,anti-miR-27a-3p and si-LncRNA RMST were transfected into H9C2 cells and added.The medium containing 200μmol/L H_(2)O_(2)was intervened for 2 h,which were recorded as si-NC+200μmol/L H_(2)O_(2)group,si-LncRNA RMST+200μmol/L H_(2)O_(2)group,miR-NC+200μmol/L H_(2)O_(2)group,miR-27a-3p+200μmol/L H_(2)O_(2)group,anti-miR-NC+si-LncRNA RMST+200μmol/L H_(2)O_(2)group,anti-miR-27a-3p+si-LncRNA RMST+200μmol/L H_(2)O_(2)group.qRT-PCR method was used to detect the expression of LncRNA RMST and miR-27a-3p.The level of LDH in the cell culture fluid,as well as the levels of MDA and SOD in the cells were tested by using kits.Flow cytometry was used to detect the rate of apoptosis.The dual luciferase reporter experiment was used to detect the targeting relationship between LncRNA RMST and miR-27a-3p.Western blot method was used to detect the expression of Cleaved-caspase-3 and Bax protein.Compared with the si-NC+200μmol/L H_(2)O_(2)group,the levels of MDA and LDH in the si-LncRNA RMST+200μmol/L H_(2)O_(2)group were decreased(P<0.05),the rate of apoptosis and the protein levels of Cleaved-caspase-3 and Bax were decreased(P<0.05),while the level of SOD was increased(P<0.05).LncRNA RMST could target the expression of miR-27a-3p.Compared with the miR-NC+200μmol/L H_(2)O_(2)group,the levels of MDA and LDH in the miR-27a-3p+200μmol/L H_(2)O_(2)group were decreased(P<0.05),the rate of cell apoptosis and the protein levels of Cleaved-caspase-3,Bax were decreased(P<0.05),while the level of SOD was increased(P<0.05).Compared with the anti-miR-NC+si-LncRNA RMST+200μmol/L H_(2)O_(2)group,the levels of MDA and LDH in the anti-miR-27a-3p+si-LncRNA RMST+200μmol/L H_(2)O_(2)group were increased(P<0.05),the apoptosis rate and the protein levels of Cleaved-caspase-3 and Bax were increased(P<0.05),while the level of SOD was decreased(P<0.05).Interfering with the expression of LncRNA RMST could inhibit the oxidative stress response and apoptosis of cardiomyocytes by targeting miR-27a-3p,thereby reducing the damage of cardiomyocytes induced by H_(2)O_(2).