miR-125b-5p对过氧化氢诱导的视网膜神经节细胞损伤的影响及其机制研究

Effect and mechanism of MiR-125b-5p on the retinal ganglion cell from injury induced by H2O2

目的研究miR-125b-5p对H2O2诱导的小鼠视网膜神经节细胞(RGCs)损伤的影响及潜在的机制。方法将RGC-5分为12组:对照组(细胞未处理组)、H2O2组(200μmol·L^-1H2O2处理12 h建立氧化损伤模型)、miRNC组、miR-125-5p组、anti-miR-NC组、anti-miR-125-5p组、H2O2+miR-NC组、H2O2+miR-125b-5p组、H2O2+si-NC组、H2O2+si-LCN2组、H2O2+miR-125b-5p+pcD NA组和H2O2+miR-125b-5p+pcD NA-LCN2组。H2O2+转染组:细胞转染miR-125b-5p或si-LCN248 h后,用200μmol·L^-1H2O2处理12 h。以免疫印迹法检测细胞中miR-125b-5p和LCN2蛋白表达水平,以流式细胞术和免疫印迹法分别检测凋亡率及其凋亡相关的B细胞淋巴瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达水平,可见分光光度法检测丙二醛(MDA)和超氧化物歧化酶(SOD)的水平。结果(1)对照组和H2O2组的miR-125b-5p蛋白表达水平分别为1.01±0.09,0.39±0.04;这2组的LCN2蛋白表达水平分别为0.43±0.04,0.87±0.08;这2组的MDA含量分别为(5.06±0.49),(15.24±1.37)nmol·mg^-1;这2组的SOD水平分别为(3.79±0.35),(0.56±0.05)U·mg^-1;这2组的细胞凋亡率分别为(7.25±0.73)%,(27.69±2.86)%;这2组的Bax蛋白表达水平分别为0.22±0.03,0.63±0.06;这2组的Bcl-2蛋白表达水平分别为0.77±0.07,0.34±0.03,组间比较,上述指标的差异均有统计学意义(均P<0.05)。(2)转染miR-125b-5p后,H2O2+miR-NC组和H2O2+miR-125b-5p组的miR-125b-5p蛋白表达水平分别为0.39±0.04,0.89±0.08;这2组的Bcl-2蛋白表达水平分别为0.34±0.03,0.64±0.06;这2组的Bax蛋白表达水平分别为0.64±0.06,0.35±0.03;这2组的细胞凋亡率分别为(29.67±2.86)%,(12.65±1.46)%;这2组的MDA含量分别为(15.47±1.36),(8.36±0.79)nmol·mg^-1;这2组的SOD水平分别为(0.48±0.04),(3.15±0.31)U·mg^-1,组间比较,上述指标的差异均有统计学意义(均P<0.05)。(3)过表达miR-125b-5p和LCN2后,H2O2+miR-125b-5p+pcD NA组和H2O2+miR-125b-5p+pcD NA-LCN2组的LCN2蛋白表达水平分别为0.37±0.04,0.74±0.07;这2组的Bcl-2蛋白表达水平分别为0.67±0.05,0.44±0.04;这2组的Bax蛋白表达水平分别为0.27±0.03,0.49±0.04;这2组的细胞凋亡率分别为(11.69±1.27)%,(22.45±2.08)%;这2组的MDA含量分别为(8.83±0.85),(12.24±1.27)nmol·mg^-1;这2组的SOD水平分别为(3.29±0.31),(1.02±0.09)U·mg^-1,组间比较,上述指标的差异均有统计学意义(均P<0.05)。结论miR-125b-5p通过靶向调控LCN2水平,减轻H2O2对RGCs的损伤并抑制细胞凋亡。

Objective injury induced by H2O2.Methods(treated with 200μmol·L^-1 H2O2 for 12 h to establish oxidative injury model),miR-NC group,miR-125-5 p group,anti-miR-NC group,anti-miR-125-5 p group,H2O2+miR-NC group,H2O2+miR-125 b-5 p group,H2O2+si-NC group,H2O2+si-LCN2 group,H2O2+miR-125 b-5 p+pcDNA group,and H2O2+miR-125 b-5 p+pcDNA-LCN2 group.H2O2+transfection group:treated with 200μmol·L^-1 H2O2 for 12 h after transfection with miR-125 b-5 p or si-LCN2 for 48 h.The expression of miR-125 b-5 p and lipocalin 2(LCN2)proteins in cells were detected by Western blot.Flow cytometry and Western blot were applied to detrmine the apoptotic rate and the expression of B-cell lymphoma-2(Bcl-2)and BCL2-Associated X(Bax),respectively.The levels of malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by visible spectrophotometry.Results ssion of miR-125 b-5 p protein in control group and H2O2 group were 1.01±0.09,0.39±0.04;the expression of LCN2 protein in the two groups were 0.43±0.04,0.87±0.08;the levels of MDA in the two groups were(5.06±0.49),(15.24±1.37)nmol·mg-1;the levels of SOD in the two groups were(3.79±0.35),(0.56±0.05)U·mg-1;the apoptosis rate in the two groups were(7.25±0.73)%,(27.69±2.86)%;the expression of Bax protein in the two groups were 0.22±0.03,0.63±0.06;the expression of Bcl-2 protein in the two groups were 0.77±0.07,0.34±0.03.Comparison between H2O2 group and control group,the difference of the factors were significant(all P<0.05).(2)After transfection with miR-125 b-5 p,the expression of miR-125 b-5 p protein in H2O2+miR-NC group and H2O2+miR-125 b-5 p group were 0.39±0.04,0.89±0.08;the expression of Bcl-2 protein in the two groups were 0.34±0.03,0.64±0.06;the expression of Bax protein in the two groups were0.64±0.06,0.35±0.03;the apoptosis rate in the two groups were(29.67±2.86)%,(12.65±1.46)%;the levels of MDA in the two groups were(15.47±1.36),(8.36±0.79)nmol·mg^-1;the levels of SOD in the two groups were(0.48±0.04),(3.15±0.31)U·mg^-1.Comparing between H2O2+miR-125 b-5 p group and H2O2+miR-NC group,the difference of the factors were significant(all P<0.05).(3)After the simultaneous overexpression of miR-125 b-5 p and LCN2,the expression of LCN2 protein in H2O2+miR-125 b-5 p+pcDNA group and H2O2+miR-125 b-5 p+pcDNA-LCN2 group were 0.37±0.04,0.74±0.07;the expression of Bcl-2 protein in the two groups were 0.67±0.05,0.44±0.04;the expression of Bax protein in the two groups were 0.27±0.03,0.49±0.04;the apoptosis rate in the two groups(11.69±1.27)%,(22.45±2.08)%;the levels of MDA in the two groups were(8.83±0.85),(12.24±1.27)nmol·mg-1;the levels of SOD in the two groups were(3.29±0.31),(1.02±0.09)U·mg-1.Comparing between H2O2+miR-125 b-5 p+pcDNA-LCN2 group and H2O2+miR-125 b-5 p+pcDNA group,the difference of the factors were significant(all P<0.05).Conclusion miR-125 b-5 p reduced the injury of retinal ganglion cells RGC-5 induced by H2O2 and inhibited apoptosis by targeting LCN2.