Silencing of MiR-23a Attenuates Hydrogen Peroxide (HO) Induced Oxidative Damages in ARPE-19 Cells by Upregulating GLS1: an in Vitro Study

Overview

Journal Cytotechnology

Specialties Biotechnology
Genetics

Date 2020 Oct 30

PMID 33123932

Citations 2

Authors

Yang Zhou

Meilibanu Yusufu

Ting Zhang

Jing Wang

Affiliations

Soon will be listed here.

Abstract

Background: Oxidative damages contributes to age-related macular degeneration (AMD) caused vision blindness, but the molecular mechanisms are still largely unknown.

Objectives: This study managed to investigate this issue by conducting in vitro experiments.

Methods: Oxidative stress were evaluated by L-012 dye, DHE staining and MDA assay. CCK-8 and colony formation assay were conducted to examine cell proliferation. Cell death was evaluated by trypan blue staining and Annexin V-FITC/PI double staining method through flow cytometry (FCM). The binding sites of miR-23a and GLS1 mRNA were predicted by online miRDB database and validated by dual-luciferase reporter gene system. Real-Time qPCR for miR-23a levels and Western Blot for protein expressions.

Results: The retinal pigment epithelial (RPE) cells (ARPE-19) were subjected to hydrogen peroxide (HO) stimulation to simulate AMD progression in vitro, and we identified a novel miR-23a/glutaminase-1 (GLS1) pathway that regulated HO induced oxidative damages in ARPE-19 cells. Mechanistically, HO induced oxidative stress, inhibited cell proliferation and induced cell death in ARPE-19 cells in a dose- and time-dependent manner. Also, HO stimulation hindered cell invasion, migration and glutamine uptake in ARPE-19 cells. Interestingly, we proved that HO increased miR-23a levels, while downregulated glutaminase-1 (GLS1) in ARPE-19 cells, and miR-23a targeted 3' untranslated region (3'UTR) of GLS1 mRNA for GLS1 degradation. Finally, our data suggested that silencing miR-23a upregulated GLS1 to reverse the detrimental effects of HO treatment on ARPE-19 cells.

Conclusions: In general, analysis of the data suggested that miR-23a ablation upregulated GLS1 to attenuate HO stimulation induced oxidative damages in ARPE-19 cells in vitro, and this study broadened our knowledge in this field, which might help to provide novel theranostic signatures for AMD.

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