miR-182 inhibited oxidative stress induce apoptosis in HLEB-3 cells by targeting cyclin dependent kinase 1

Objective To investigate the protective mechanism of miRNA-182 on human lens epithelial cells (HLE-B3) apoptosis induced by oxidative stress during cataract formation. Methods Human lens epithelium cells (HLE-B3) were stimulated by H2O2(100 μmol/L, 12 h) and the expression of miR-182 was evaluated using qRT-PCR. The expression of CDK1 was determined by qRT-PCR and western blot. To confirm our hypothesis, we analyzed miR-182 and CDK1 gene expression in the lens anterior capsules of patients with and without cataracts by qRT-PCR. Luciferase report assay was used to confirm the target gene of miR-182. The changes in apoptosis related protein (Bax, Bad, caspase-3) and target gene CDK1 protein were detected by Western bloting. Results While CDK1 expression was suppressed, a significant increase was observed in the expression of miR-182 in both human cataract lens anterior capsular samples and HLE-B3 cell lines stimulated by H2O2 compared with normal control group. Over-expression of miR-182 significantly suppressed H2O2-induced HLE-B3 cell apoptosis in vitro, a critical step in cataract formation. The expression of miR-182 was also decreased in cataract lens anterior capsular tissues and H2O2-induced HLE-B3 cell lines. nockdown of miR-182 significantly up-regulated the expression of CDK1 at the level of mRNA and protein. Luciferase report assay showed CDK1 was a direct target gene of miR-182. After 12 hours of pre-transfection of miR-182 followed by another 12 hours of oxidative stress stimulation, the expression of CDK1 was down-regulated. In contrast, the expression of CDK1 was promoted by miR-182-inhibitor in the same condition. The transfection of miR-182 could reverse apoptosis process in H2O2 induced HLE-B3 cells through down-regulated of expressions of apoptosis-related proteins Bax, Bad and caspase-3. Conclusion We demonstrated for the first time that miR-182 is a functional gene of cataract and knockdown of miR-182 reduced the expression of CDK1. These results suggest that miR-182/CDK1/Rb/P16INK4a signaling pathway is a novel mechanism for promoting cataract formation. Key words: Cataract; Oxidative stress; miR-182; CDK1; Apoptosis