Role of microRNA-146a in regulation of fibrosis in orbital fibroblasts from patients with Graves' orbitopathy

Aim: To examine the role of microRNA-146a (miR-146a) in the regulation of fibrosis in an in vitro model of Graves' orbitopathy (GO). Methods: Orbital fat/connective tissues were harvested from patients with GO and non-GO for primary orbital fibroblast cultures. The effects of transforming growth factor-β (TGF-β), a potent cytokine that promotes fibrosis, on miR-146a expression were analysed in GO and non-GO orbital fibroblasts using quantitative real-time PCR. The effects of overexpressed miR-146a on TGF-β-induced fibrotic markers were examined in GO orbital fibroblasts by western blot analysis. Expression of Sma and Mad related family (Smad) 4/tumour necrosis factor receptor-associated factor 6 (TRAF6) after transfection of miR-146a mimics or inhibitors were examined. Results: TGF-β induced an increase in miR-146a expression in orbital fibroblasts from patients with GO in a time-dependent and concentration-dependent manner. miR-146a mimics further decreased the production of TGF-β-induced fibronectin, collagen Iα and α-smooth muscle actin protein. The Smad4 and TRAF6 protein levels were significantly decreased by miR-146a mimics, compared with control mimics, and significantly increased on inhibition of miR-146a production compared with a control. Conclusions: miR-146a plays a role as a negative regulator in the production of TGF-β-induced fibrotic markers. Thus, miR-146a may be involved in the regulation of fibrosis in orbital fibroblasts from patients with GO.