LINC00473调控miR-210/TET2分子轴对H2O2诱导的人晶状体上皮细胞生物学特性的影响

Effect of LINC00473 on biological characteristics of H2O2 induced human lens epithelial cells by regulating miR-210/TET2 molecular axis

目的探讨LINC00473调控miR-210/10-11易位酶2(TET2)分子轴对H2O2诱导的人晶状体上皮细胞生物学特性的影响。方法人晶状体上皮细胞SRA01/04传代培养后,采用100μmol·L^-1 H2O2处理24 h。将SRA01/04细胞分为Con组、H2O2组、H2O2+pcDNA3.1组、H2O2+pcDNA3.1-LINC00473组、H2O2+pcDNA3.1-LINC00473+miR-NC组、pcDNA3.1-LINC00473+miR-210组、H2O2+pcDNA3.1-LINC00473+si-NC组、H2O2+pcDNA3.1-LINC00473+si-TET2组。细胞计数试剂盒(CCK-8)检测细胞活力,流式细胞术检测细胞凋亡,试剂盒检测细胞中丙二醛(MDA)含量和超氧化物歧化酶(SOD)、谷光甘肽过氧化物酶(GSH-Px)活性。双荧光素酶报告基因实验检测LINC00473与miR-210、miR-210与TET2的靶向结合关系。结果Con组与H2O2组、H2O2+pcDNA3.1组与H2O2+pcDNA3.1-LINC00473组、H2O2+pcDNA3.1-LINC00473+miR-NC组与pcDNA3.1-LINC00473+miR-210组、H2O2+pcDNA3.1-LINC00473+si-NC组与H2O2+pcDNA3.1-LINC00473+si-TET2组相比,细胞存活率、细胞凋亡率、克隆形成数、MDA含量、SOD和GSH-Px活性差异均有统计学意义(均为P<0.05)。双荧光素酶报告基因实验结果显示,miR-NC和WT-LINC00473共转染组SRA01/04细胞荧光素酶活性(0.36±0.03)较miR-210 mimics和WT-LINC00473共转染组(0.96±0.10)显著降低(P<0.05);miR-NC和WT-TET2共转染组SRA01/04细胞的荧光素酶活性(0.33±0.03)较miR-210 mimics和WT-TET2共转染组(0.94±0.09)显著降低,差异有统计学意义(P<0.05);LINC00473靶向作用miR-210并下调其表达水平,miR-210可负调控TET2的表达水平。结论LINC00473通过下调miR-210/TET2分子轴减轻对H2O2对人晶状体上皮细胞增殖、凋亡和氧化损伤的影响。

Objective To investigate the effect of LINC00473 on the biological characteristics of H2O2 induced human lens epithelial cells by regulating miR-210/10-11 transposase 2(TET2)molecular axis.Methods Human lens epithelial SRA01/04 cells were cultured with 100μmol·L^-1 H2O2 for 24 h.SRA01/04 cells were divided into Con group,H2O2 group,H2O2+pcDNA3.1 group,H2O2+pcDNA3.1-LINC00473 group,H2O2+pcDNA3.1-LINC00473+miR-NC group,pcDNA3.1-LINC00473+miR-210 group,H2O2+pcDNA3.1-LINC00473+si-NC group,and H2O2+pcDNA3.1-LINC00473+si-TET2 group.Cell counting kit-8(CCK-8)was used to detect cell viability;flow cytometry was used to detect cell apoptosis;the kit was used to detect malondialdehyde(MDA)content,activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px).The dual luciferase reporter gene assay detected the targeted binding correlation of LINC00473 with miR-210,of miR-210 with TET2.Results Statistical differences were observed in cell viability,cell apoptosis rate,colony forming efficiency,MDA content,activities of SOD and GSH-Px for Con group comparing with H2O2 group,H2O2+pcDNA3.1 group comparing with H2O2+pcDNA3.1-LINC00473 group,H2O2+pcDNA3.1-LINC00473+miR-NC group comparing with pcDNA3.1-LINC00473+miR-210 group,H2O2+pcDNA3.1-LINC00473+si-NC group comparing with H2O2+pcDNA3.1-LINC00473+si-TET2 group(all P<0.05).The dual luciferase reporter gene confirmed that SRA01/04 cell luciferase activity was 0.36±0.03 in miR-NC and WT-LINC00473 cotransfection group,and obviously lower than 0.96±0.10 in miR-210 mimics and WT-LINC00473 cotransfection group(P<0.05);SRA01/04 cell luciferase activity was 0.33±0.03 in miR-NC and WT-TET2 cotransfection group,and obviously lower than 0.94±0.09 in miR-210 mimics and WT-TET2 cotransfection group(P<0.05).LINC00473 targeted miR-210 and down-regulated its expression level,and miR-210 negatively regulated the expression level of TET2.Conclusion LINC00473 can reduce the effect of H2O2 on proliferation,apoptosis and oxidative damage of human lens epithelial cells by down-regulating miR-210/TET2 molecular axis.