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

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

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。

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-21 mimics 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-PI3 K 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-PI3 K 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.