miR-24对过氧化氢诱导的HLE-B3细胞凋亡的影响及其与线粒体Smac/Diablo-XIAP-caspase-9/3凋亡途径的关系

Effect of miR-24 on hydrogen peroxide-induced apoptosis of HLE-B3 cells and its correlation with Smac/Diablo-XIAP-caspase-9/3 pathway

目的探讨微小RNA\|24(miR-24)对过氧化氢(H_(2)O_(2))诱导的HLE-B3细胞凋亡的影响,分析其与线粒体中第二个线粒体衍生的胱天蛋白酶激活因子/低等电位点的凋亡抑制蛋白的直接结合蛋白(the second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI,Smac/Diablo)-XIAP-caspase-9/3凋亡途径的关系。方法将HLE-B3细胞分为对照组、H_(2)O_(2)组、H_(2)O_(2)+miR-24 NC组和H_(2)O_(2)+miR-24抑制剂组。先按照miR-24抑制剂、miR-24 NC试剂盒说明书转染H_(2)O_(2)+miR-24抑制剂组和H_(2)O_(2)+miR-24 NC组细胞24 h,然后向H_(2)O_(2)组、H_(2)O_(2)+miR-24 NC组和H_(2)O_(2)+miR-24抑制剂组HLE-B3细胞中加入200μmol·L^(-1)H_(2)O_(2)干预24 h,收集细胞用于后续实验;将正常培养的HLE-B3细胞设置为对照组。采用实时荧光定量PCR检测各组HLE-B3细胞miR-24表达情况,CCK-8法检测各组HLE-B3细胞生长情况,用流式细胞仪检测各组细胞凋亡情况,计算各组细胞存活率和细胞凋亡率。采用荧光探针JC-1法检测线粒体膜电位变化情况(利用红绿荧光强度比反映线粒体膜电位);采用蛋白免疫印迹法检测各组HLE-B3细胞线粒体和细胞质分级中Smac/Diablo及细胞质分级中XIAP、caspase-9和caspase-3蛋白表达情况。对所得数据进行统计学分析。结果当H_(2)O_(2)浓度大于200μmol·L^(-1)时,细胞存活率均小于50%,本实验以200μmol·L^(-1)作为最适H_(2)O_(2)浓度。对照组、H_(2)O_(2)组、H_(2)O_(2)+miR-24 NC组和H_(2)O_(2)+miR-24抑制剂组细胞miR-24相对表达量分别为1.04±0.02、2.73±0.09、2.69±0.07和1.15±0.06;与对照组相比,H_(2)O_(2)组细胞中miR-24表达升高(P<0.05);与H_(2)O_(2)组和H_(2)O_(2)+miR-24 NC组相比,H_(2)O_(2)+miR-24抑制剂组中miR-24表达降低(均为P<0.05),但H_(2)O_(2)组与H_(2)O_(2)+miR-24 NC组中miR-24表达差异无统计学意义(P>0.05)。与H_(2)O_(2)组和H_(2)O_(2)+miR-24 NC组相比,H_(2)O_(2)+miR-24抑制剂组细胞存活率显著升高、细胞凋亡率显著降低(均为P<0.05),但H_(2)O_(2)组与H_(2)O_(2)+miR-24 NC组细胞存活率和细胞凋亡率差异均无统计学意义(均为P>0.05)。对照组、H_(2)O_(2)组、H_(2)O_(2)+miR-24 NC组和H_(2)O_(2)+miR-24抑制剂组红绿荧光强度比分别为0.24±0.02、0.12±0.02、0.15±0.03、0.31±0.06。与对照组相比,H_(2)O_(2)组红绿荧光强度比降低(P<0.05);与H_(2)O_(2)组和H_(2)O_(2)+miR-24 NC组相比,H_(2)O_(2)+miR-24抑制剂组红绿荧光强度比均升高(均为P<0.05),但H_(2)O_(2)组与H_(2)O_(2)+miR-24 NC组红绿荧光强度比比较,差异无统计学意义(P>0.05)。与H_(2)O_(2)组和H_(2)O_(2)+miR-24 NC组相比,H_(2)O_(2)+miR-24抑制剂组线粒体分级中Smac/Diablo蛋白及细胞质分级中XIAP蛋白表达升高(均为P<0.05),细胞质分级中Smac/Diablo、caspase-9和caspase-3蛋白表达降低(均为P<0.05),但H_(2)O_(2)组与H_(2)O_(2)+miR-24 NC组线粒体、细胞质分级中Smac/Diablo蛋白及细胞质分级中XIAP、caspase-9和caspase-3蛋白表达相比,差异均无统计学意义(均为P>0.05)。结论miR-24可能通过抑制线粒体Smac/Diablo-XIAP-caspase-9/3凋亡途径,抑制H_(2)O_(2)诱导的HLE-B3细胞凋亡。

Objective To explore the effect of microRNA-24(miR-24)on apoptosis of human lens epithelial cells(HLE-B3)induced by hydrogen peroxide(H_(2)O_(2)),and to analyze its relationship with mitochondrial Smac/Diablo-XIAP-caspase-9/3 apoptosis pathway.Methods HLE-B3 cells were divided into control group,H_(2)O_(2)group,H_(2)O_(2)+miR-24 NC group and H_(2)O_(2)+miR-24 inhibitor group.Cells in H_(2)O_(2)+miR-24 inhibitor group and H_(2)O_(2)+miR-24 NC group were transfected for 24 h according to the miR-24 inhibitor and miR-24 NC kit instructions,and then 200μmol·L^(-1)H_(2)O_(2)was added to the cells in the H_(2)O_(2)group,H_(2)O_(2)+miR-24 NC group and H_(2)O_(2)+miR-24 inhibitor group for 24 h,and the cells were collected for subsequent experiments.The normally cultured HLE-B3 cells were set as the control group.Real-time fluorescent quantitative PCR method was used to detect the expression of miR-24 in HLE-B3 cells;CCK-8 method was used to detect the growth of HLE-B3 cells in each group;flow cytometry was used to detect the apoptosis of HLE-B3 cells,and finally,cell survival rate and cell apoptosis rate were calculated.Fluorescent probe JC-1 method was used to detect the change of mitochondrial membrane potential(The ratio of red-green fluorescence intensity can reflects the mitochondrial membrane potential).Western blot was used to detect the expression of the second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI(Smac/Diablo),XIAP,caspase-9 and caspase-3 proteins.Statistical analysis was performed on the obtained data.Results When the H_(2)O_(2)concentration was greater than 200μmol·L^(-1),the cell survival rate was less than 50%,so in this experiment,200μmol·L^(-1)was used as the optimal H_(2)O_(2)concentration.The relative expression level of miR-24 in the control group,H_(2)O_(2)group,H_(2)O_(2)+miR-24 NC group and H_(2)O_(2)+miR-24 inhibitor group was 1.04±0.02,2.73±0.09,2.69±0.07 and 1.15±0.06,respectively;compared with control group,the expression level of miR-24 in H_(2)O_(2)group was significantly higher,and the difference was statistically significant(P<0.05);compared with the H_(2)O_(2)group and the H_(2)O_(2)+miR-24 NC group,the expression of miR-24 in the H_(2)O_(2)+miR-24 inhibitor group was lower,and the difference was statistically significant(both P<0.05);however,the expression of miR-24 in the H_(2)O_(2)group and the H_(2)O_(2)+miR-24 NC group was not significantly different(P>0.05).Compared with the H_(2)O_(2)group and the H_(2)O_(2)+miR-24 NC group,the cell survival rate in the H_(2)O_(2)+miR-24 inhibitor group was increased and the cell apoptosis rate was decreased,and the differences were statistically significant(both P<0.05);however,there was little difference in cell survival rate and apoptosis rate between H_(2)O_(2)group and H_(2)O_(2)+miR-24 NC group(both P>0.05).The red-green fluorescence intensity ratio in the control group,H_(2)O_(2)group,H_(2)O_(2)+miR-24 NC group and H_(2)O_(2)+miR-24 inhibitor group was 0.24±0.02,0.12±0.02,0.15±0.03 and 0.31±0.06,respectively.Compared with the control group,the red-green fluorescence intensity ratio in the H_(2)O_(2)group decreased,and the difference was statistically significant(P<0.05);compared with H_(2)O_(2)group and H_(2)O_(2)+miR-24 NC group,the red-green fluorescence intensity ratio in H_(2)O_(2)+miR-24 inhibitor group increased,and the difference was statistically significant(P<0.05);however,the red-green fluorescence intensity ratio between H_(2)O_(2)group and H_(2)O_(2)+miR-24 NC group changed little,and the difference was not statistically significant(P>0.05).Compared with the H_(2)O_(2)group and the H_(2)O_(2)+miR-24 NC group,the expression of Smac/Diablo protein in the mitochondrial grading and XIAP protein in the cytoplasmic grading in the H_(2)O_(2)+miR-24 inhibitor group increased,and the expression of Smac/Diablo,caspase-9 and caspase-3 proteins in cytoplasmic grading decreased,and the differences were statistically significant(all P<0.05);however,the expression of Smac/Diablo protein in mitochondria and cytoplasmic grading,XIAP,caspase-9 and caspase-3 proteins in cytoplasmic grading in H_(2)O_(2)group and H_(2)O_(2)+miR-24 NC group changed little,and the differences were not statistically significant(all P>0.05).Conclusion MiR-24 may inhibit the H_(2)O_(2)-induced apoptosis of HLE-B3 cells by inhibiting mitochondrial Smac/Diablo-XIAP-caspase-9/3 apoptosis pathway.