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

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。

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

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 100 μmol·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+ anti-miR-155-5p+si-SEP15 group (all P<0.05). Conclusion　miR-155-5p can relieve the injury of HLE-B3 induced by H₂O₂ and inhibit apoptosis by targeting SEP15 genes.