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

目的　探讨微小RNA-24(miR-24)对过氧化氢（H₂O₂）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、H₂O₂组、H₂O₂+miR-24 NC组和H₂O₂+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H₂O₂+miR-24抑制剂组和H₂O₂+miR-24 NC组细胞24 h，然后向H₂O₂组、H₂O₂+miR-24 NC组和H₂O₂+miR-24 抑制剂组HLE-B3细胞中加入200 μmol·L^-1 H₂O₂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H₂O₂浓度大于200 μmol·L^-1时，细胞存活率均小于50%，本实验以200 μmol·L^-1 作为最适H₂O₂浓度。对照组、H₂O₂组、H₂O₂+miR-24 NC组和H₂O₂+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，H₂O₂组细胞中miR-24表达升高（P<0.05）；与H₂O₂组和H₂O₂+miR-24 NC组相比，H₂O₂+miR-24抑制剂组中miR-24表达降低（均为P<0.05），但H₂O₂组与H₂O₂+miR-24 NC组中miR-24表达差异无统计学意义（P>0.05）。与H₂O₂组和H₂O₂+miR-24 NC组相比，H₂O₂+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H₂O₂组与H₂O₂+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、H₂O₂组、H₂O₂+miR-24 NC组和H₂O₂+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，H₂O₂组红绿荧光强度比降低（P<0.05）；与H₂O₂组和H₂O₂+miR-24 NC组相比，H₂O₂+miR-24抑制剂组红绿荧光强度比均升高（均为P<0.05），但H₂O₂组与H₂O₂+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H₂O₂组和H₂O₂+miR-24 NC组相比，H₂O₂+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H₂O₂组与H₂O₂+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H₂O₂诱导的HLE-B3细胞凋亡。

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

Objective　To explore the effect of microRNA-24 (miR-24) on apoptosis of human lens epithelial cells (HLE-B3) induced by hydrogen peroxide (H₂O₂), and to analyze its relationship with mitochondrial Smac/Diablo-XIAP-caspase-9/3 apoptosis pathway.Methods　HLE-B3 cells were divided into control group, H₂O₂ group, H₂O₂+miR-24 NC group and H₂O₂+miR-24 inhibitor group. 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. Results　When the H₂O₂ concentration was greater than 200 μmol·L^-1, the cell survival rate was less than 50%, so in this experiment, 200 μmol·L^-1 was used as the optimal H₂O₂ concentration. The relative expression level of miR-24 in the control group, H₂O₂ group, H₂O₂+miR-24 NC group and H₂O₂+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 H₂O₂ group was significantly higher, and the difference was statistically significant (P<0.05); compared with the H₂O₂ group and the H₂O₂+miR-24 NC group, the expression of miR-24 in the H₂O₂+miR-24 inhibitor group was lower, and the difference was statistically significant (both P<0.05); however, the expression of miR-24 in the H₂O₂ group and the H₂O₂+miR-24 NC group was not significantly different (P>0.05). Compared with the H₂O₂ group and the H₂O₂+miR-24 NC group, the cell survival rate in the H₂O₂+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 H₂O₂ group and H₂O₂+miR-24 NC group (both P>0.05). The red-green fluorescence intensity ratio in the control group, H₂O₂ group, H₂O₂+miR-24 NC group and H₂O₂+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 H₂O₂ group decreased, and the difference was statistically significant (P<0.05); compared with H₂O₂ group and H₂O₂+miR-24 NC group, the red-green fluorescence intensity ratio in H₂O₂+miR-24 inhibitor group increased, and the difference was statistically significant (P<0.05); however, the red-green fluorescence intensity ratio between H₂O₂ group and H₂O₂+miR-24 NC group changed little, and the difference was not statistically significant (P>0.05). Compared with the H₂O₂ group and the H₂O₂+miR-24 NC group, the expression of Smac/Diablo protein in the mitochondrial grading and XIAP protein in the cytoplasmic grading in the H₂O₂+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 H₂O₂ group and H₂O₂+miR-24 NC group changed little, and the differences were not statistically significant (all P>0.05).Conclusion　MiR-24 may inhibit the H₂O₂-induced apoptosis of HLE-B3 cells by inhibiting mitochondrial Smac/Diablo-XIAP-caspase-9/3 apoptosis pathway.