miR-155-5p靶向SEP15基因在H2O2诱导的人晶状体上皮细胞损伤中的作用  

Effect of miR-155-5p on H2O2-induced human lens epithelial cell injury by targeting SEP15 genes

作　　者：张虹 陈颖平 陈圣文[1] ZHANG Hong;CHEN Yingping;CHEN Shengwen(Department of Ophthalmology,the Second Affiliated Hospital of Hainan Medical College,Haikou 570216,Hainan Province,China)

机构地区：[1]海南医学院第二附属医院眼科,海南省海口市570216

出　　处：《眼科新进展》2020年第5期425-429,共5页Recent Advances in Ophthalmology

摘　　要：目的探讨miR-155-5p靶向SEP15基因对H2O2诱导的人晶状体上皮细胞损伤的影响及潜在机制。方法用含100μmol·L^-1 H2O2的培养液培养人晶状体上皮细胞系HLE-B3建立H2O2损伤模型,根据处理和转染情况分为对照组、H2O2组、H2O2+anti-miR-NC组、H2O2+anti-miR-155-5p组、H2O2+pcDNA组、H2O2+pcDNA-SEP15组、miR-NC组、miR-155-5p组、H2O2+anti-miR-155-5p+si-NC组、H2O2+anti-miR-155-5p+si-SEP15组,Real-time PCR检测HLE-B3细胞中miR-155-5p和SEP15 mRNA的表达,Western blot测定SEP15、Bcl-2、Bax和Cleaved-caspase-3蛋白的表达,ELISA测定H2O2诱导后HLE-B3中丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)水平,流式细胞术测定细胞凋亡率,Targetscan预测miR-155-5p与SEP15的结合关系,双荧光素酶检测验证miR-155-5p与SEP15的调控关系。结果H2O2组HLE-B3细胞中miR-155-5p、SEP15 mRNA、SEP15蛋白、MDA、SOD、GSH-Px含量及凋亡率与对照组相比差异均有统计学意义(均为P<0.05)。H2O2+anti-miR-155-5p组和H2O2+anti-pcDNA-SEP15组的细胞凋亡率分别为(12.69±1.28)%和(14.67±1.52)%,分别与H2O2+anti-miR-NC组的(25.68±2.34)%和H2O2+pcDNA组的(23.65±2.17)%相比,差异均有统计学意义(均为P<0.05)。Targetscan预测miR-155-5p与SEP15存在结合位点,双荧光素酶检测结果显示,miR-155-5p组野生型WT-SEP-15的萤火虫荧光素酶相对活性为0.39±0.03,较miR-NC组的1.02±0.09显著降低(P=0.000)。与H2O2+anti-miR-155-5p-si-NC组相比,H2O2+anti-miR-155-5p+si-SEP15组的SEP-15蛋白、Bcl-2蛋白、SOD活性和GSP-Px含量均显著降低,Bax蛋白、MDA含量及细胞凋亡率均显著升高(均为P<0.05)。结论miR-155-5p通过靶向SEP15基因以减轻H2O2对人晶状体上皮细胞HLE-B3的损伤并抑制细胞凋亡。Objective To investigate the effects of miR-155-5p on H2O2-induced human lens epithelial cell(HLE-B3 cells)injury and its potential mechanisms.Methods HLE-B3 cells were cultured with 100μmol·L^-1 H2O2 to establish H2O2 injury models.According to the treatment and transfection conditions,HLE-B3 cells were divided into control group,H2O2 group,H2O2+anti-miR-NC group,H2O2+anti-miR-155-5p group,H2O2+pcDNA group,H2O2+pcDNA-SEP15 group,miR-NC group,miR-155-5p group,H2O2+anti-miR-155-5p+si-NC group,and H2O2+anti-miR-155-5p+si-SEP15 group.The expression levels of miR-155-5p and SEP15 mRNA were detected by Real-time PCR.Protein expression of SEP15,Bcl-2,Bax and Cleaved-caspase-3 were determined by Western blot.The levels of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in H2O2-induced HLE-B3 cells were measured by ELISA.The apoptosis of HLE-B3 cells was determined by flow cytometry.The binding relationship between miR-155-5p and SEP15 was predicted by Targetscan.The relationship between miR-155-5p and SEP15 was verified by dual-luciferase reporter assay system.Results Statistical differences were found in contents of miR-155-5p,SEP15 mRNA,SEP15 protein,MDA,SOD,GSH-Px and apoptosis rate of HLE-B3 cells between H2O2 group and control group(all P<0.05).The apoptosis rates were(12.69±1.28)%and(14.67±1.52)%in H2O2+anti-miR-155-5p group and H2O2+anti-pcDNA-SEP15 group,which had statistical differences with(25.68±2.34)%in H2O2+anti-miR-NC group and(23.65±2.17)%in H2O2+pcDNA group(all P<0.05).Targetscan predicted that there was a binding site between miR-155-5p and SEP15.Results of the dual-luciferase reporting system indicated that relative activity of firefly luciferase of wild-type WT-SET-15 was 0.39±0.03 in miR-155-5p group,which was lower than 1.02±0.09 in miR-NC group(P=0.000).Compared with H2O2+anti-miR-155-5p+si-NC group,levels of SEP-15 protein,Bcl-2 protein,SOD activity and GSP-Px content were obviously lower,and Bax protein,MDA content and apoptosis rate were higher in H2O2+ant

关 键 词：人晶状体上皮细胞 HLE-B3 miR-155-5p SEP15 细胞损伤 细胞凋亡 

分 类 号：R776[医药卫生—眼科]