miR-125b对晶状体上皮细胞的抗氧化应激作用及其机制

Anti-oxidative stress effects of miR-125b on lens epithelial cells and its mechanism

目的探讨微小RNA 125b(miR-125b)对年龄相关性白内障晶状体上皮细胞(LECs)抗氧化应激能力的影响及其可能的作用机制。方法收集2018年7月至2019年3月于河南省立眼科医院接受白内障超声乳化手术的年龄相关性白内障患者24例24眼的晶状体前囊膜组织标本24份,同期纳入河南省立眼科医院眼库的20例20眼供体正常前囊膜组织20份,分别采用逆转录PCR和Western blot法检测并比较不同标本LECs中miR-125b和核因子E2相关因子2(Nrf2)的表达量;将人LECs细胞系HLEB-3细胞分为对照组和氧化应激模型组,采用不同浓度的H2O2(100、200、400 μmol/L)共培养细胞建立氧化应激细胞模型,对照组采用不含H2O2的培养基。细胞培养24 h后采用DCFH-DA荧光探针测定不同浓度H2O2处理组细胞内源性活性氧簇(ROS)含量,ELISA法检测总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)浓度;分别采用逆转录PCR和Western blot法检测各组细胞中miR-125b和Nrf2的表达水平。用含有miR-125b拟似物、miR-125b对照和miR-125b抑制物序列的质粒分别转染HLEB-3细胞24 h,采用DCFH-DA荧光探针法测定并比较不同转染组细胞中ROS含量,ELISA法检测T-AOC、SOD和GSH-Px活性及MDA浓度;采用双荧光素酶报告法验证miR-125b与Nrf2的靶向关系;采用Western blot法检测各转染组细胞内Nrf2蛋白表达水平;采用免疫荧光双染色法检测不同转染组细胞中Nrf2和Keap1的表达和定位。结果正常晶状体前囊膜组织中miR-125b和Nrf2的相对表达量分别为0.21±0.03和0.27±0.06,少于白内障前囊膜组织中的0.89±0.05和0.84±0.12,差异均有统计学意义(t=15.355,P<0.05;t=18.647,P<0.05)。各浓度H2O2处理组细胞中miR-125b和Nrf2相对表达量均明显高于对照组,miR-125b和Nrf2相对表达量随着H2O2浓度的增加而升高,差异均有统计学意义(均P<0.05);与对照组比较,各浓度H2O2处理组细胞内T-AOC、SOD和GSH-Px活性均明显降低,ROS含量和MDA浓度均明显升高,差异均有统计学意义(均P<0.05);与miR-125b对照组比较,miR-125b拟似物组细胞中T-AOC、SOD和GSH-Px活性均明显升高,ROS含量和MDA浓度均明显降低,差异均有统计学意义(均P<0.05);miR-125b抑制物组细胞中T-AOC、SOD和GSH-Px活性均明显低于miR-125b对照组,ROS含量和MDA浓度均明显高于miR-125b对照组,差异均有统计学意义(均P<0.05)。双荧光素酶报告结果提示H2O2刺激后细胞中Nrf2发生不同程度的核转移,miR-125b拟似物组细胞质中Nrf2荧光最强,核转移也最显著,细胞质中Keap1的表达微弱;miR-125b抑制物组细胞质中Nrf2荧光强度最弱,核转移少,细胞质中Keap1荧光表达增强。结论 miR-125b能增强年龄相关性白内障LECs的抗氧化应激能力,其机制可能与miR-125b靶向刺激Nrf2表达上调,进而调控Keap1/Nrf2信号通路活性有关。

Objective To investigate the anti-oxidative stress effects of microRNA 125b(miR-125b)on lens epithelial cells(LECs)and its possible mechanism.Methods Twenty-four anterior capsule specimens were collected from 24 eyes of 24 age-related cataract patients during phacoemulsification and 20 normal anterior capsule specimens were obtained from 20 eyes of 20 donors in Henan Eye Hospital from July 2018 to March 2019 under the approval of a Medical Ethics Committee of Henan Eye Hospital(No.YKYY20193151).The reverse transcription PCR and Western blot assay were employed to detect and compare the relative expression levels of miR-125b and nuclear factor E2-related factor 2(Nrf2)in different specimens.The human lens epithelial cell line HLEB-3 was divided into control group and oxidative stress model group.The oxidative stress models were established by coculture with different concentrations(100,200,400μmol/L)of H2O2 for 24 hours,and the cells were cultured with normal medium without H2O2 in the control group.The reactive oxygen species(ROS)content was detected by DCFH-DA fluorescent probe,and the activities of total-antioxidative capability(T-AOC),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)as well as malondialdehyde(MDA)concentration were detected by ELISA,and compared among the groups.The expression levels of miR-125b and Nrf2 were detected by reverse transcription PCR and Western blot assay,respectively.The cells were transfected with miR-125b mimics,miR-125b control and miR-125b inhibitor for 24 hours,respectively,and ROS content was detected by DCFH-DA fluorescent probe and T-AOC,SOD and GSH-Px activities as well as MDA concentration were detected by ELISA and compared among different transfected groups.A dual luciferase reporter assay was used to assess an association between miR-125b and Nrf2.The expression level of Nrf2 protein was detected by Western blot assay and the expression levels of Nrf2 and Keap1 were assayed and located by immunofluorescence double staining.Results The relative expression levels of miR-125b and Nrf2 in the normal lens anterior capsule specimens were 0.21±0.03 and 0.27±0.06,which were significantly lower than 0.89±0.05 and 0.84±0.12 in the cataract specimens,respectively(t=15.355,P<0.05;t=18.647,P<0.05).The relative expression levels of miR-125b and Nrf2 were significantly increased in various H2O2 treated groups in comparison with the control group and were gradually elevated with the increase of H2O2 concentration(all at P<0.05).Compared with the control group,the T-AOC,SOD and GSH-Px activities were reduced,and ROS content and MDA concentration were significantly ascended(all at P<0.05).Compared with the miR-125b control group,the T-AOC,GSH-Px and SOD activities were increased,and ROS content and MDA concentration were decreased in the miR-125b mimics group(all at P<0.05).In addition,the T-AOC,GSH-Px and SOD activities were significantly weakened,and ROS content and MDA concentration were significantly increased in the miR-125b inhibitor group in comparison with the miR-125b control group(all at P<0.05).Dual luciferase reporter assay showed that miR-125b targeted to the expression of Nrf2 in the H2O2 model cells.The fluorescence of Nrf2 in the cytoplasm was the strongest with more nuclear transfer in the miR-125b mimics group,and the expression intensity of Keap1 in the cytoplasm was weaker.The expression of Nrf2 was the weakest with less nuclear transfer in the miR-125b inhibitor group,and the expression level of Keap1 in the cytoplasm was stronger.Conclusions MiR-125b can enhance the anti-oxidative stress of LECs in age-related cataractous eyes probably by upregulating the expression of Nrf2 and activating the Keap1/Nrf2 signaling pathway.