Inhibitory effect of microRNA-23b-3p on high glucose-induced autophagy and apoptosis of human lens epithelial cells and its mechanism / 中华实验眼科杂志

ABSTRACT

Objective:To investigate the regulatory effects of microRNA-23b-3p (miR-23b-3p) on the autophagy and apoptosis of human lens epithelial cells induced by high glucose.Methods:Thirty diabetic cataract (DC) patients as DC group and 30 patients with simple cataract as simple cataract group were enrolled in The First Affiliated Hospital of Xi'an Medical University from September 2019 to October 2020.Conventional phacoemulsification and intraocular lens transplantation were performed in both groups.The anterior capsular tissue was collected during the operation.The expression of miR-23b-3p in the anterior lens capsule was detected by real-time fluorescence quantitative PCR (RT-qPCR). Human lens epithelial cell line HLEB3 cells were cultured in vitro and divided into normal control group and high-glucose group, which were cultured in normal and high-glucose medium, respectively.The targeting relationship between proto-cadherin 17 (PCDH17) and miR-23b-3p was predicted according to the bioinformatics database, and was verified by the dual-luciferase reporter gene experiment.High glucose-cultured HLEB3 cells were divided into miR-23b-3p mimics group, negative control (NC) mimics group, NC-siRNA group, PCDH17-siRNA group, miR-23b-3p mimics+ Vector group, miR-23b-3p mimics+ pcDNA-PCDH17 group, and were transfected with corresponding reagents according to grouping.The expression of miR-23b-3p and PCDH17 mRNA was detected by RT-qPCR.The expressions of a mammalian homolog of yeast Atg6/Vps30 (Beclin-1), microtubule-associated protein 1 light chain 3 (LC3B), c-Jun N-terminal kinases (JNK), phosphorylated (p-) JNK, c-Jun, p-c-Jun, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) proteins were assayed by Western blot.The apoptosis rate was detected by flow cytometry.The study protocol was approved by the Ethics Committee of The First Affiliated Hospital of Xi'an Medical College (No.LSL2019037). Written informed consent was obtained from each patient. Results:The relative expression of miR-23b-3p in the anterior lens capsule of DC group was 0.35±0.15, which was significantly lower than 1.00±0.09 of simple cataract group ( t=44.627, P<0.01). There were significant differences in the relative expression levels of miR-23b-3p, LC3B Ⅱ/Ⅰ, Beclin-1, Bcl-2 and Bax proteins among normal control group, high glucose group, high glucose+ NC mimics group and high glucose+ miR-23b-3p mimics group ( F=21.325, 28.318, 17.634, 15.482, 22.325, 26.537; all at P<0.01). Compared with normal control group, the apoptosis rate, LC3B Ⅱ/Ⅰ, Beclin-1 and Bax protein expressions in high glucose group were significantly increased, and the Bcl-2 protein expression was significantly decreased (all at P<0.05). Compared with NC mimics group, the apoptosis rate, LC3B Ⅱ/Ⅰ, Beclin-1, and Bax protein expressions were significantly decreased and the Bcl-2 protein expression was significantly increased in miR-23b-3p mimics group (all at P<0.05). The results of bioinformatics and dual-luciferase reporter gene experiments showed that PCDH17 was a target gene of miR-23b-3p, and the relative expression of PCDH17 mRNA in miR-23b-3p mimics group was significantly lower than that in NC mimics group ( P<0.05). Compared with NC-siRNA group, the apoptosis rate, LC3B Ⅱ/Ⅰ, Beclin-1 and Bax protein expressions in PCDH17-siRNA group were significantly decreased, and the Bcl-2 protein expression was significantly increased ( t=9.116, 12.413, 5.349, 3.273, 8.419; all at P<0.01). There were significant differences in the relative expression levels of p-JNK/JNK, p-c-Jun/c-Jun, LC3B Ⅱ/Ⅰ, Beclin-1, Bcl-2 and Bax proteins in NC mimics group, miR-23b-3p mimics group, miR-23b-3p mimics+ Vector group and miR-23b-3p mimics+ pcDNA-PCDH17 group ( F=24.724, 19.319, 23.418, 17.562, 20.263, 15.249; all at P<0.05). Compared with the miR-23b-3p mimics+ Vector group, the expressions of p-JNK/JNK, p-c-Jun/c-Jun, LC3B Ⅱ/Ⅰ, Beclin-1 and Bax were significantly increased, and the expression of Bcl-2 protein was decreased in miR-23b-3p mimics+ pcDNA-PCDH17 group (all at P<0.05). Conclusions:MiR-23b-3p have a protective effect on HLEB3 cells in a high-glucose environment, mainly by targeting PCDH17 to regulate the JNK signaling pathway to inhibit high glucose-induced autophagy and apoptosis.