Effect of miR200a and SIRT1 expression on epithelial cell apoptosis in age-related cataract lens epithelial cells / 国际生物医学工程杂志

ABSTRACT

Objective:To investigate the effects of miR200a and SIRT1 expression on epithelial cell apoptosis in age-related cataract lens epithelial cells.Methods:30 anterior capsule samples of age-related cataract patients were enrolled from the ophthalmology outpatient department of Beichen Hospital affiliated with Nankai University from January to August 2022 as the cataract group and another 30 normal anterior capsule samples of corneal transplantation were enrolled during the same period as the control group. Real -time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression levels of miR200a and SIRT1 genes in the cataract group and control group. The oxidative stress level divided human lens epithelial cells SRA01/04 into a negative control group, miR200a overexpression group, and miR200a inhibition group. The negative control group, miR200a overexpression plasmid, and miR200a inhibitor group were transfected, respectively. Cell apoptosis rate and oxidative stress level were detected by flow cytometry, and Western Blot was used to detect the expression of SIRT1 protein and Caspase-3, Bcl-2, and Bcl-2 related X (Bax) proteins containing cysteine in each group of cells. The targeting relationship between miR200a and SIRT1 was validated using dual luciferase reporter gene detection. Results:Compared with the control group, the expression level of miR200a in the cataract group was significantly increased ( P < 0.01), while the expression level of SIRT1 was significantly reduced ( P < 0.01). Compared with the control group, the levels of SOD, GSH, and GSH-Px in the cataract group were significantly reduced (all P < 0.01), and the MDA level was significantly higher than those in the control group ( P < 0.01). The apoptosis rate of the miR200a overexpression group was significantly higher than that of the negative control group ( P < 0.05), and the apoptosis rate of the miR200a inhibition group was significantly lower than that of the negative control group, the differences were statistically significant ( P < 0.05). The level of SIRT1 protein in the miR200a overexpression group was significantly lower than that in the negative control group ( P < 0.05), and the level of SIRT1 protein in cells of the miR200a inhibition group was significantly higher than that of the negative control group ( P < 0.05). Compared with the negative control group, the miR200a overexpression group showed a significant increase in Caspase-3 activity and Bax protein (all P < 0.05), while the Bcl-2 protein level was significantly reduced ( P < 0.05). The miR200a inhibition group showed a significant decrease in Caspase-3 activity and Bax protein (all P < 0.05), while the Bcl-2 protein level was significantly increased ( P < 0.05). The dual luciferase experiment showed that the overexpression group of miR-200a can significantly inhibit the luciferase activity of the wild-type SIRT1 plasmid, which is the target gene of miR-200a. Conclusions:miR220a is highly expressed in the capsule of lens of patients with age-related cataracts, and its mechanism may be that miR220a promotes apoptosis of lens epithelial cells by targeting the expression of SIRT1.