miR-138调控年龄相关性白内障晶状体上皮细胞抗氧化应激作用的机制

摘要

·AIM:To investigate the effects and mechanism of miR-138 in mediating the antioxidant function of lens epithelial cells affected by age-related cataracts. ·METHODS:Real-time quantitative PCR(RT-qPCR) was used to detect miR-138 expression in the anterior lens capsules of healthy people, the anterior lens capsules of patients with age-related cataracts, and human epithelial cell line (SRA01/04) cells exposed to oxidative stress. A 2',7'-dichloro-fluorescein diacetate (DCFH-DA) probe was used to measure the levels of endogenous reactive oxygen species (ROS) in human lens epithelial cells (hLECs) exposed to 400μ mol/L H2O2for 1h. SRA01/04 cells were transfected with either miR-138 mimics,mimic controls, miR-138 inhibitors or inhibitor controls. After 72h,these cells were exposed to 400μ mol/L H2O2for 1h, then p53 and Bax mRNA expression were measured using RT-qPCR. Expression of p53 and Bax protein were also measured by western blotting analysis. Finally, cell viability was assessed using an MTS assay. ·RESULTS: Compared to the control group, expression of miR-138 in the anterior lens capsules of age-related cataract patients and in SRA01/04 cells exposed to oxidative stress significantly increased (P<0.001). Levels of endogenous ROS were significantly elevated in hLECs exposed to oxidative stress (P<0.001). Compared to the mimic control group, the hLECs in the miR-138 mimic group expressed significantly higher levels of p53 and Bax mRNA and protein while cell viability was significantly reduced(P<0.001). Conversely, p53 and Bax mRNA and protein expression were significantly reduced in the miR-138 inhibitor group as compared to the control group, while the cells in this group had much higher levels of cell viability (P<0.001). · CONCLUSION: The expression of miR - 138 is upregulated in the anterior lens capsules of age-related cataract patients. MiR-138 decreases the anti-oxidative stress capacity of lens epithelial cells by upregulating p53 and Bax, while inhibiting cell proliferation and repair. This finding suggests that miR-138 may play a key role in the development of age-related cataracts.%目的:探讨miR-138对年龄相关性白内障晶状体上皮细胞抗氧化应激能力的影响及其作用机制.方法:采用实时定量PCR(RT-qPCR)检测年龄相关性白内障与正常人透明晶状体前囊膜组织中及人晶状体上皮细胞系(SRA01/04)氧化应激模型中miR-138的表达水平.利用2',7'-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针检测细胞内源性活性氧(reactive oxygen species,ROS)水平.向人晶状体上皮细胞中分别转染 miR-138 mimics, mimic controls,miR-138 inhibitors,inhibitor controls 72h后细胞暴露于400μmol/L H2O21h,采用RT-qPCR检测p53和Bax的mRNA表达,western blotting检测p53和Bax的蛋白表达水平,MTS法检测细胞增殖活力.结果:与正常对照组相比,年龄相关性白内障晶状体组织中与人晶状体上皮细胞氧化应激模型中miR-138的表达均显著升高,差异有统计学意义(P<0.001);人晶状体上皮细胞氧化应激模型中内源性ROS的水平明显升高,差异有统计学意义(P<0.001).相对于对照组,miR-138 mimics组p53和 Bax 的 mRNA、蛋白表达水平均明显升高,细胞增殖活力明显下降,差异有统计学意义(均 P<0.001);miR-138 inhibitors组p53和Bax的mRNA、蛋白表达水平均明显下降,细胞增殖活力显著升高,差异有统计学意义(均P<0.001).结论:miR-138在年龄相关性白内障囊膜组织中表达上调,通过正调控下游靶基因p53和Bax,下调人晶状体上皮细胞抗氧化应激的能力,抑制人晶状体上皮细胞增殖和修复,从而参与年龄相关性白内障的发生过程.