Abstract
Purpose: :
Thyroid Eye Disease (TED) is characterized by orbital infiltration of white blood cells and accumulation of the nonsulfated glycosaminoglycan, hyaluronan (HA). HA contributes significant connective tissue expansion in TED by virtue of its extraordinary hydrophilic nature. microRNAs (miRNAs) are extensively involved in diverse biological processes. However, little is known about the role of miRNAs in regulating the production of HA in orbital tissue. We hypothesized that miRNAs would influence HA synthesis in orbital fibroblasts. In this study,we report that miR130a increases both basal and TGF-β-stimulated HA production in orbital fibroblasts. miR130a’s mechanism of action in regulating HA production may be through inhibiting the adenosine monophosphate (AMP)- activated protein kinase (AMPK) signal transduction pathway.
Methods: :
Primary orbital fibroblasts were isolated from TED patients undergoing orbital decompression surgery. The cells were grown in RPMI 1640 media containing 10% FBS. Non-specific control miR, miR130a, anti-miR130a or AMPK-β1 siRNA were introduced into orbital fibroblasts using lipofectamine. The amount of HA in the cell culture supernatant was measured by ELISA. Relative miR130a expression and HA Synthases (HAS) 1-3 mRNA expression were measured by qPCR. AMPK-β1 and AMPK-α1 protein expression were detected with western blot.
Results: :
We found miR-130a was highly expressed in TED orbital fat tissue compared to fat from other tissues. Overexpression of miR-130a significantly increased HA production both in basal and TGF-beta stimulated TED orbital fibroblasts. When miR-130a was inhibited by an anti-sense RNA, the amount of HA production decreased. Overexpression of miR-130a also significantly increased HAS1 and 2 expression both in basal and TGF-beta stimulated TED fibroblasts. Basal levels of HAS3 mRNA expression also increased, but the TGF-beta stimulated levels of HAS3 mRNA did not change significantly. We also found that one target of miR130a may be the AMPK subunit, AMPK-β1. AMPK-β1protein levels were significantly decreased by overexpression of miR130a. Furthermore, down regulation of AMPK-β1 by siRNA significantly increased HAS 1 and 2 mRNA expression and HA production in orbital fibroblasts.
Conclusions: :
Our data indicate that miR130a is involved in HA synthesis and may act via down-regulation of the AMPK signal transduction pathway. Reduction of AMPK-β1 leads to higher expression of HAS1 and HAS2 and significantly enhances the production of HA. These studies provide new insight into the role of miR130a and AMPK in the pathogenesis of TED.
Keywords: signal transduction: pharmacology/physiology • extracellular matrix • gene/expression