Silencing of miR-23a attenuates hydrogen peroxide (H 2 O 2 ) induced oxidative damages in ARPE-19 cells by upregulating
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ORIGINAL ARTICLE
Silencing of miR-23a attenuates hydrogen peroxide (H2O2) induced oxidative damages in ARPE-19 cells by upregulating GLS1: an in vitro study Yang Zhou . Meilibanu Yusufu . Ting Zhang . Jing Wang
Received: 2 February 2020 / Accepted: 16 October 2020 Ó Springer Nature B.V. 2020
Abstract Background Oxidative damages contributes to agerelated 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
Y. Zhou M. Yusufu Department of Ophthalmology, The Fifth Affiliated Hospital of Xinjiang Medical University, Henan Road No. ¨ ru¨mqi 830011, Xinjiang, China 118, U e-mail: [email protected] M. Yusufu e-mail: [email protected] T. Zhang J. Wang (&) Department of Eye Center, Qingdao Municipal Hospital (Group), Jiaozhou Road No.1, Qingdao 266011, Shandong, China e-mail: [email protected]
miR-23a levels and Western Blot for protein expressions. Results The retinal pigment epithelial (RPE) cells (ARPE-19) were subjected to hydrogen peroxide (H2O2) stimulation to simulate AMD progression in vitro, and we identified a novel miR-23a/glutaminase-1 (GLS1) pathway that regulated H2O2 induced oxidative damages in ARPE-19 cells. Mechanistically, H2O2 induced oxidative stress, inhibited cell proliferation and induced cell death in ARPE-19 cells in a dose- and time-dependent manner. Also, H2O2 stimulation hindered cell invasion, migration and glutamine uptake in ARPE-19 cells. Interestingly, we proved that H2O2 increased miR-23a levels, while downregulated glutaminase-1 (GLS1) in ARPE-19 cells, and miR-23a targeted 30 untranslated region (30 UTR) of GLS1 mRNA for GLS1 degradation. Finally, our data suggested that silencing miR-23a upregulated GLS1 to reverse the detrimental effects of H2O2 treatment on ARPE-19 cells. Conclusions In general, analysis of the data suggested that miR-23a ablation upregulated GLS1 to attenuate H2O2 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. Keywords Age-related macular degeneration Oxidative stress ARPE-19 miR-23a GLS1
T. Zhang e-mail: [email protected]
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Cytotechnology
Introduction Age-related macular degeneration (AMD) is the most frequent senile ophthalmic disease that leads to substantial vision degeneration and even blindness (O’Neill et al. 2020; Sato et al. 2019), and retinal pigment epithelial
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