Sun Young Jang1, Min Kyung Chae2, Joon H Lee3, Eun Jig Lee4, Jin Sook Yoon2. 1. Department of Ophthalmology Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Bucheon, Korea 2Department of Medicine, Yonsei University Graduate School of Medicine, Seoul. 2. Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea. 3. Myung-Gok Eye Research Institute at Kim's Eye Hospital, Konyang University College of Medicine, Nonsan, Korea. 4. Department of Endocrinology, Severance Hospital, Institue of Endocrine Research, Yonsei University College of Medicine, Seoul, Korea.
Abstract
PURPOSE: To investigate the role of microRNA 146a (miR-146a) in the regulation of inflammation in an in vitro model of Graves' orbitopathy (GO). METHODS: The level of miR-146a expression in orbital adipose tissue was compared between GO and non-GO by quantitative real-time PCR (qPCR). The effects of interleukin 1β (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 nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen-activated protein kinase/extracellular signal-regulated kinases (MEK)-1/2, c-Jun N-terminal kinases (JNK)-1/2, p38 MAP kinase, and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) 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 = 0.032). Interleukin 1β induced a time- and concentration-dependent increase in miR-146a expression. Interleukin 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-κB, 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 with IL-1β-induced miR-146 expression, independent t-test). Interleukin 1β-induced IL-6 protein production was further decreased by miR-146a mimics, but not by inhibitors of miR-146a. CONCLUSIONS: MicroRNA 146a was upregulated by inflammatory stress in orbital fibroblasts. Our results indicated that miR-146a had a positive effect on the anti-inflammatory process. MicroRNA 146a may play a role in the regulation of inflammation in orbital fibroblasts, and may participate in the pathogenesis of GO.
PURPOSE: To investigate the role of microRNA 146a (miR-146a) in the regulation of inflammation in an in vitro model of Graves' orbitopathy (GO). METHODS: The level of miR-146a expression in orbital adipose tissue was compared between GO and non-GO by quantitative real-time PCR (qPCR). The effects of interleukin 1β (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 nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen-activated protein kinase/extracellular signal-regulated kinases (MEK)-1/2, c-Jun N-terminal kinases (JNK)-1/2, p38 MAP kinase, and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) 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 = 0.032). Interleukin 1β induced a time- and concentration-dependent increase in miR-146a expression. Interleukin 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-κB, 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 with IL-1β-induced miR-146 expression, independent t-test). Interleukin 1β-induced IL-6 protein production was further decreased by miR-146a mimics, but not by inhibitors of miR-146a. CONCLUSIONS:MicroRNA 146a was upregulated by inflammatory stress in orbital fibroblasts. Our results indicated that miR-146a had a positive effect on the anti-inflammatory process. MicroRNA 146a may play a role in the regulation of inflammation in orbital fibroblasts, and may participate in the pathogenesis of GO.
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