Hsa-MiR-483 -3p regulates the extracellular matrix proteins via TGFβ2/SMAD4 signaling in the glucocorticoid-responsive human trabecular meshwork cells.

Abstract

The purpose of this study was to investigate the role of hsa-miR-483-3p on the regulation of extracellular matrix (ECM) in cultured human trabecular meshwork (HTM) cells with known steroid response. Primary cultures of HTM cells with known steroid responsiveness [GC-responder (GC-R) and GC-Non-responder (GC-NR) cells] were grown on coverslip in 12-well plate until 80% confluence and treated with 100 nM dexamethasone (DEX) for 24 h and transfected with different concentrations of synthetic miRNA 483-3p mimic or inhibitor. After 24 h or 72 h post transfection, the cells were harvested for the following experiments: (i) percentage transfection efficiency, (ii) RNA isolation for qPCR analysis, (iii) immunofluorescence staining, and (iv) protein isolation for Western blotting, respectively. All experiments were performed in triplicate with three biological samples (n = 3). GC-R HTM cells showed significantly higher expression of SMAD4 as compared to GC-NR HTM cells. Similarly, DEX treatment up-regulated the SMAD4-dependent ECM proteins. The presence of a miR-483-3p mimic down-regulated SMAD4 expression and SMAD4-dependent ECM production in a dose-dependent manner by negatively down-regulating SMAD4/TGF-β2 signaling. The inhibition of SMAD4-dependent ECM production by miR-483-3p was more pronounced in GC-R HTM cells as compared to GC-NR cells. The down-regulation in ECM production by miR-483-3p is SMAD4-dependent and may play a protective role in mitigating steroid response in GC-R HTM cells. Using miR-483-3p mimics demonstrates therapeutic potential for the management of steroid-induced ocular hypertension and glaucoma.