Promoting effect of miR-15a on high glucose-induced oxidative stress in human lens epithelial cells and its mechanism

Authors: Nan Anchao,  Nan Jianru,  Liu Yadong,  Liu Xiangling
DOI: 10.3760/cma.j.cn115989-20200301-00126
Published 2022-05-10
Cite asChin J Exp Ophthalmol, 2022, 40(5): 422-430.

Abstract

Objective

To investigate the effect of microRNA-15a (miR-15a) on the anti-oxidative stress ability of human lens epithelial cells (LECs) induced by high glucose and its possible mechanism.

Methods

The anterior lens capsule specimens from patients with diabetic cataract (DC) and healthy donors were collected.The expressions of miR-15a and silent information regulator 1 (SIRT1) in the specimens were detected by real-time quantitative PCR (RT-qPCR) and Western blot, respectively.The human lens epithelial cell line HLEB-3 cells were cultured with 0, 10, 20, or 50 mmol/L glucose for 24 hours.The expression of miR-15a in the cells was detected by RT-qPCR.The expressions of SIRT1, forkhead transcription factor 3a (FOXO3a), and p53 proteins in the cells were determined by Western blot.The cell apoptosis was assayed by flow cytometry.The endogenous reactive oxygen species (ROS) content in the cells was measured by 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA). The total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) activities in the cells were identified.HLEB-3 cells were transfected with miR-15a control or miR-15a inhibitor, then incubated with 50 mmol/L glucose for 24 hours.Cell apoptosis of the transfected cells was detected by flow cytometry.The endogenous ROS expression in the transfected cells was determined by DCFH-DA.The T-AOC, SOD, and GSH-Px activities as well as MDA concentration were measured.The relationship between miR-15a and SIRT1 was verified by dual-luciferase reporter assay.The SIRT1, FOXO3a, and p53 protein expressions in the transfected cells were detected by Western blot.This study was approved by an Ethics Committee of the Second Affiliated Hospital of Zhengzhou University (No.ZDEFY201803160023). Written informed consent was obtained from each subject.

Results

The relative expression of miR-15a in normal lens anterior capsule tissue was 0.21±0.02, which was lower than 0.96±0.10 in lens anterior capsule tissue of DC patients, and the difference was statistically significant (t=12.231, P<0.001). The relative expression of SIRT1 in the anterior lens capsule tissue was 0.89±0.09, which was higher than 0.31±0.05 in the anterior lens capsule tissue of DC patients, showing a statistically significant difference (t=8.964, P<0.001). With the increase of glucose concentration, the relative expression of miR-15a, FOXO3a, and p53 in cells increased, and the relative expression of SIRT1 decreased; the apoptosis rate of cells increased; the ROS content and MDA concentration increased; the activities of T-AOC, SOD and GSH-Px decreased, and the differences were statistically significant (all at P<0.05). The apoptosis rate, ROS content, and MDA concentrations were higher in miR-15a control group than miR-15a inhibitor group, and the activities of T-AOC, SOD, and GSH-Px were lower in miR-15a control group than miR-15a inhibitor group, with statistically significant differences (all at P<0.05). The luciferase activity of the SIRT1-3′-untranslated region (UTR)-wild type (WT) reporter gene in miR-15a control group was significantly lower than that in miR-15a inhibitor group, and the difference was statistically significant (t=5.978, P=0.004). No significant difference was found in the luciferase activity of the SIRT1-3′-UTR-mutant type (MUT) reporter gene (t=0.432, P=0.688). The relative expressions of FOXO3a and p53 proteins were significantly higher in miR-15a control group than miR-15a inhibitor group, and the relative expression of SIRT1 protein was significantly lower in miR-15a control group than miR-15a inhibitor group, showing statistically significant differences (all at P<0.05).

Conclusions

miR-15a can inhibit the anti-oxidative stress damage ability of LECs induced by high glucose, which may be achieved by inhibiting the expression of SIRT1 to up-regulate the activities of FOXO3a and p53, and aggravating apoptosis.

Key words:

Oxidative stress; Cataract, diabetic; Epithelial cells, lens; microRNA-15a; Silent information regulator 1; Apoptosis

Figures and tables

Contributor Information

Nan Anchao

Department of Ophthalmology, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, China

Nan Jianru

Department of Ophthalmology, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, China

Liu Yadong

Department of Ophthalmology, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, China

Liu Xiangling

Department of Ophthalmology, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, China

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