miRNA-21-5p对光诱导的人视网膜色素上皮细胞氧化应激损伤的影响

Effect of micro ribonucleic acid-21-5p on light-induced oxidative stress injury in human retinal pigment epithelial cells

目的研究miRNA-21-5p对光诱导的人视网膜色素上皮细胞氧化应激损伤的影响。方法将体外培养的人视网膜色素上皮细胞系ARPE-19细胞随机分为对照组(TransIntro EL Transfection Reagent转染液培养)、损伤组(TransIntro EL Transfection Reagent转染液+光损伤)、过表达组(TransIntro EL Transfection Reagent转染液+miRNA-21-5p mimics+光损伤)、阴性组(TransIntro EL Transfection Reagent转染液+miRNA-21-5p mimics NC+光损伤)、PI3K/Akt阻断剂组(TransIntro EL Transfection Reagent转染液+miRNA-21-5p mimics+LY294002+光损伤)。使用光照强度为(16500±200)lx的LED冷光灯建立ARPE-19细胞光损伤模型,利用TransIntro EL Transfection Reagent转染液行细胞转染。采用qRT-PCR法检测各组ARPE-19细胞miRNA-21-5p表达水平,采用CCK-8法检测各组ARPE-19细胞活力,流式细胞仪检测各组ARPE-19细胞活性氧(ROS)含量变化,ELISA法检测各组ARPE-19细胞超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果与对照组相比,损伤组ARPE-19细胞miRNA-21-5p表达明显下降,细胞存活率明显下降,ROS含量显著升高,SOD活性明显降低,MDA含量明显增加(均为P<0.001);与损伤组相比,过表达组ARPE-19细胞miRNA-21-5p表达明显升高,细胞存活率明显上升,ROS含量明显降低,SOD活性升高,MDA含量减少(均为P<0.001),而阴性组ARPE-19细胞miRNA-21-5p表达、细胞存活率、ROS含量、SOD活性、MDA含量均无明显差异(均为P>0.05);与过表达组相比,PI3K/Akt阻断剂组ARPE-19细胞miRNA-21-5p表达明显降低,细胞存活率明显下降,ROS含量明显升高,SOD活性明显降低,MDA含量明显增加(均为P<0.01)。结论miRNA-21-5p能显著降低光诱导的ARPE-19细胞氧化应激水平,提高光诱导的ARPE-19细胞抗氧化能力。

Objective To investigate the effect of micro ribonucleic acid(miRNA)-21-5p on light-induced oxidative stress injury in human retinal pigment epithelial cells.Methods Adult human retinal pigment epithelial(ARPE)-19 cells cultured in vitro were randomly divided into the control group(TransIntro EL Transfection Reagent transfection),damage group(TransIntro EL Transfection Reagent transfection+optical damage),overexpression group(TransIntro EL Transfection Reagent transfection+miRNA-21-5p mimics+optical damage),negative group(TransIntro EL Transfection Reagent transfection+miRNA-21-5p mimics NC+optical damage),and PI3K/Akt blocker group(TransIntro EL Transfection Reagent transfection+miRNA-21-5p mimics+LY294002+optical damage).The optical damage model of ARPE-19 cells was established with a cold-light LED lamp with an illumination intensity of(16500±200)lx,and the cells were transfected with TransIntro EL Transfection Reagent.The expression level of miRNA-21-5p was detected by quantitative reverse transcription PCR;the viability of ARPE-19 cells was detected by Cell Counting Kit-8;the changes of reactive oxygen species(ROS)in ARPE-19 cells were detected by flow cytometry.The viability of superoxide dismutase(SOD)and level of malondialdehyde(MDA)were detected by enzyme-linked immunosorbent assay.Results Compared with the control group,the miRNA-21-5p expression,survival rate and SOD viability of ARPE-19 cells significantly decreased,and the levels of ROS and MDA significantly increased in the damage group(all P<0.001).Compared with the damage group,the miRNA-21-5p expression,survival rate and SOD viability of ARPE-19 cells significantly increased,and the levels of ROS and MDA significantly reduced in the overexpression group(all P<0.001),while there were no significant differences in these parameters in the negative group(all P>0.05).Compared with the overexpression group,the miRNA-21-5p expression,survival rate and SOD viability of ARPE-19 cells significantly decreased,and the levels of ROS and MDA significantly increased in the PI3K/Akt blocker group(all P<0.01).Conclusion The miRNA-21-5p significantly reduces the level of light-induced oxidative stress and improves the light-induced antioxidant capacity of ARPE-19 cells.