miR-21对H2O2诱导的视网膜色素上皮细胞凋亡及PTEN/AKT通路的影响

目的　探讨miR-21对H₂O₂诱导的视网膜色素上皮细胞凋亡以及PTEN/AKT通路的影响。方法　体外培养人视网膜色素上皮细胞系ARPE-19，实验分为四组:空白对照组、阴性对照组、H₂O₂组、H₂O₂+miR-21mimics组，使用q-RT-PCR法检测各组ARPE-19细胞中miR-21表达，流式细胞术检测各组细胞凋亡率，Western blot检测凋亡相关蛋白Bax、Bcl-2、Caspase-3以及人第10号染色体缺失的磷酸酶（phosphatase and tensin homolog deleted on chromosome ten，PTEN）、磷酸化AKT蛋白表达。结果　与空白对照组和阴性对照组相比，H₂O₂组和H₂O₂+miR-21 mimics组miR-21表达量（1.14±0.23、2.18±0.44）、SOD水平［（5.49±1.10） U·L^-1、（14.28±2.86） U·L^-1］、Bcl-2蛋白含量（0.34±0.07、0.62±0.12）、p-PI3K表达水平（0.46±0.09、0.68±0.13）、p-AKT水平（0.53±0.11、1.16±0.13）均显著降低，差异均有统计学意义（均为P＜0.05），而活性氧自由基（reactive oxygen species,ROS）水平（30.58±7.96、23.25±5.67）、MDA水平［（3.95±0.79）mol·L^-1、（2.13±0.43）mol·L^-1］、凋亡率［（25.48±5.10）%、（17.63±3.52）%］、PTEN蛋白表达（0.59±0.12、0.43±0.11）、Bax表达（0.73±0.15、0.49±0.10）、Caspase-3蛋白表达（0.85±0.17、0.42±0.08）均显著升高，差异均有统计学意义（均为P＜0.05）。与H₂O₂组相比，H₂O₂+miR-21 mimics组miR-21表达量、SOD水平、Bcl-2蛋白表达、p-PI3K及p-AKT蛋白表达均显著升高（均为P＜0.05），ROS水平、MDA水平、细胞凋亡率、PTEN蛋白表达、Bax及Caspase-3蛋白表达均显著降低（均为P＜0.05）。结论　上调miR-21可能通过激活PTEN/AKT信号通路抑制H₂O₂诱导的视网膜色素上皮细胞细胞凋亡。

Effects of MiR-21 on apoptosis of retinal pigment epithelial cells induced by oxidative stress and PTEN/AKT pathway

Objective　To investigate the effects of miR-21 on the apoptosis of retinal pigment epithelial cells induced by hydrogen peroxide and the PTEN/AKT pathway.Methods　The human retinal pigment epithelial cell line ARPE-19 was cultured in vitro,which were divided into four groups:blank control group,negative control group,H₂O₂ group,H₂O₂+miR-21mimics group.The qR-PCR method was used to detect the miR-21 of each group of ARPE-19 cells.Flow cytometry was used to detect the apoptotic rate of ARPE-19 cells in each group.The expression levels of apoptotic cell-associated protein (BCL2-associated X,Bax),B-cell lymphoma-2 (Bcl-2),Caspase-3 and phosphatase,tensin homolog deleted on chromosome ten (PTEN) and phosphorylated AKT proteins were detected by Western blot.Results　The expression levels of miR-21 in the H₂O₂ group and the H₂O₂+miR-21 mimics group were 1.14±0.23 and 2.18±0.44,respectively,and the SOD levels were （5.49±1.10）U·L^-1 and （14.28±2.86）U·L^-1,respectively,the protein content of Bcl-2 was 0.34±0.07 and 0.62±0.12,respectively,and the expression levels of p-PI3K were 0.46±0.09 and 0.68±0.13,respectively,and the p-AKT levels were 0.53±0.11 and 1.16±0.13,respectively,which were decreased significantly compared with the blank group and the negative control group (all P<0.05).The ROS levels were 30.58±7.96 and 23.25±5.67,respectively,the MDA levels were (3.95±0.79)mol·L^-1 and (2.13±0.43)mol·L^-1,respectively,the apoptotic rates were (25.48±5.10)%,(17.63± 3.52)%,respectively,PTEN protein expression was 0.59±0.12,0.43±0.11,Bax was 0.73±0.15,0.49±0.10,and Caspase 3 protein levels were 0.85±0.17 and 0.42±0.08,respectively,which were increased significantly compared with the blank group and the negative control group (all P<0.05).Compared with H₂O₂ group,the expression of miR-21,SOD,Bcl-2,p-PI3K and p-AKT were significantly increased in H₂O₂+miR-21 mimics group ( all P<0.05),ROS level,MDA level,apoptotic rate,PTEN protein expression,Bax,cleaved-Caspase 3 protein levels were significantly decreased (all P<0.05).Conclusion　Up-regulation of miR-21 may inhibit H₂O₂-induced apoptosis of retinal pigment epithelial cells by activating PTEN/AKT signaling pathway.