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 ofSma 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.
- experimental laboratory
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Contributors SYJ and JSY were responsible for the study conception and design, as well as the intellectual content of the paper. SJP made intellectual contributions to the text. MKC performed experiments. JHL and EJL revised the article critically for intellectual content. All authors read and approved the final manuscript.
Funding This study was supported by a faculty research grant from Yonsei University, College of Medicine (grant no. 6-2015-0046 to JSY) and by the National Research Foundation of Korea (NRF-2017R1A1A1A05001051).
Competing interests None declared.
Patient consent Obtained.
Ethics approval This study was approved by the Institutional Review Board of the Severance Hospital, Yonsei University College of Medicine.
Provenance and peer review Not commissioned; externally peer reviewed.