Role of miR-146a in the regulation of inflammation in an in vitro model of Graves’ orbitopathy

Abstract

Purpose: To investigate the role of miR-146a in the regulation of inflammation in an in vitro model of Graves’ orbitopathy (GO). Materials and Methods: The orbital adipose tissues of GO patients (n=16) and control group (n=12) were used in this study. The level of miR-146a expression was compared between GO and control group by quantitative real-time PCR (qPCR). The effects of IL-1β on miR-146a expression were analyzed in orbital fibroblasts by qPCR. To investigate the molecular mechanism underlying IL-1β-induced miR-146a expression, the effects of inhibitors of NF-B, MEK-1/2, JNK-1/2, p38 MAP kinase, and PI3-K were analyzed. The effects of miR-146a mimics and inhibitors on IL-1β-induced IL-6 release were examined by ELISA and Western blotting. Results: The level of miR-146a expression was significantly higher in GO orbital adipose tissue than in non-GO (p<0.001). IL-1β induced a time- and concentration-dependent increase in miR-146a expression. IL-1β (10 ng/mL, 16 hours) induced an approximately 17.5-fold increase in miR-146 expression. The increase in miR-146a expression by IL-1β was significantly inhibited by NF-B, JNK-1/2, and PI3K inhibitors (1.94 ± 0.25, 5.28 ± 0.34 and 9.73 ± 2.32-fold, respectively, p<0.05 compared to IL-1β-induced miR-146 expression, independent t test). IL-1β-induced IL-6 protein production was further decreased by miR-146a mimics, but not by inhibitors of miR-146a. Conclusions: miR-146a was upregulated by inflammatory stress in orbital fibroblasts. Our results indicated that miR-146a had a positive effect on the anti-inflammatory process. miR-146a may play a role in the regulation of inflammation in orbital fibroblasts, and may participate in the pathogenesis of GO.