Role Of Mir-30b On The Regulation Of The Catalase Gene In Human ARPE-19 Cells

Abstract

Purpose: : Oxidative stress (OS) from reactive oxygen species has been implicated in many diseases, including age-related macular degeneration, in which the RPE is considered a primary target. Alterations in miRNA expression may occur following exposure to several stress-inducing agents, including hydrogen peroxide (H₂O₂). The aim of the present study was to investigate the miR-30-mediated regulation of catalase expression in response to H₂O₂ treatment in ARPE-19.

Methods: : ARPE-19 cells were maintained in DMEM/F-12 medium supplemented with 10% FBS, penicillin (100U/ml) and streptomycin (100ug/ml) at 37 °C in a humidified environment containing 5% CO₂. In order to study the effect of OS on the regulation of miRNA and catalase expression, cells were treated with H₂O₂ for 18 hrs. Targeting of miR-30b to the catalase 3'UTR was evaluated using the pmirGLO luciferase system (Promega). Catalase expression was also measured in ARPE-19 cells transfected with miR-30b and miR-30d mimics and antagomirs. Using miRNA-enriched total RNA, qRT-PCR was performed to determine the relative expression level of miRs and catalase.

Results: : H₂O₂ treatment increased expression of Dicer, miR-30b and miR-30d, indicative of the possible involvement of H₂O₂ in the modulation of miR-30 expression. The mimics of miR-30b significantly reduced luciferase expression in ARPE-19 cells cotransfected with the catalase 3'UTR-luc reporter construct compared to the control (scrambled mimics). The decrease in luciferase activity by the miR-30b-mimics was significantly prevented when the miR-30b antagomirs were used. Downregulation of catalase gene transcript by miR-30b-mimics as confirmed by qRT-PCR was also disinhibited by mir-30b-antagomirs that resulted in enhanced catalase expression. However, compared with the control, mimics of miR-30d had no effect on catalase expression.

Conclusions: : miR-30b negatively regulates catalase expression and its expression is responsive to H₂O₂-mediated oxidative stress. Antagomir-mediated inhibition of miR-30b contributed to increased catalase expression. Therefore, using a miRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system.

Keywords: age-related macular degeneration • retinal pigment epithelium • oxidation/oxidative or free radical damage