Abstract
BackgroundMicroRNAs (miRNAs) are abnormally expressed in various ocular diseases, including age-related cataract. However, the role of miR-182-5p in the progression of age-related cataract remains unclear.MethodsThe expression of miR-182-5p in HLE-B3 cells was detected by qRT-PCR. HLE-B3 cells were transfected with miR-182-5p mimics. CCK-8, EdU, flow cytometry, 2′,7′-dichlorodihydrofluorescein diacetate, JC-1 kit, and western blot were used to assess the cell viability, proliferation, apoptosis, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. The relationship between miR-182-5p and NOX4 was confirmed using the dual-luciferase reporter gene analysis.ResultsWe found that miR-182-5p expression was significantly decreased by the H2O2 exposure. Overexpression of miR-182-5p promoted cell proliferation and inhibited ROS production and apoptosis in H2O2-induced HLE-B3 cells. Moreover, p-p-38, p-ERK, and p-JNK were up-regulated in H2O2-treated HLE-B3 cells, and overexpression of miR-182-5p reversed the effects of H2O2 on HLE-B3 cells. In addition, dual-luciferase reporter assay substantiated that NOX4 was a direct target and downregulated by miR-182-5p.ConclusionsWe concluded that miR-182-5p inhibited lens epithelial cells apoptosis through regulating NOX4 and p38 MAPK signaling, providing a novel biomarker for treatment of age-related cataract.
Highlights
MicroRNAs are abnormally expressed in various ocular diseases, including age-related cataract
Oxidative damage to the human lens epithelial cells (LECs) is one of the major factors leading to apoptosis which is considered as an early event of cataract development [7, 8]
The results showed that nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (NOX4) overexpression reversed the inhibition of apoptosis induced by miR-182-5p mimics in H2O2-treated Human lens epithelial B3 (HLE-B3) cells (Fig. 6D)
Summary
Cell culture Human lens epithelial B3 (HLE-B3) cells were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). Cell transfection MiR-182-5p mimics or negative controls (RiboBio, Guangzhou, China) were transfected into HLE-B3 cells using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. At 24, 48, 72 and 96 h, 10 μL of CCK8 reagent (Beyotime Institute of Biotechnology, Jiangsu, China) was added to the cells. The collected cells were resuspended in 500 μL of 1× binding buffer, 5 μL Annexin V-FITC and 5 μL PI were added and incubated at room temperature in the dark for 15 min. Detection of mitochondrial membrane potential (MMP) Cells were added to 6-well plates (1 × 106) and divided into groups as described for cell transfection. The changes of cell MMP in different groups of cells were measured using 5 μg/mL JC-1 (Beyotime Biotechnology, Shanghai, China). Differences between groups were considered significant when P < 0.05
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