TGF-β–Mediated Downregulation of MicroRNA-196a Contributes to the Constitutive Upregulated Type I Collagen Expression in Scleroderma Dermal Fibroblasts

Abstract

Previous reports indicated the significance of the TGF-β signaling in the pathogenesis of systemic sclerosis. We tried to evaluate the possibility that microRNAs (miRNAs) play a part in the type I collagen upregulation seen in normal fibroblasts stimulated with exogenous TGF-β and systemic sclerosis (SSc) fibroblasts. miRNA expression profile was evaluated by miRNA PCR array and real-time PCR. The protein expression of type I collagen was determined by immunoblotting. In vivo detection of miRNA in paraffin section was performed by in situ hybridization. Several miRNAs were found to be downregulated in both TGF-β-stimulated normal fibroblasts and SSc fibroblasts compared with normal fibroblasts by PCR array. Among them, miR-196a expression was decreased in SSc both in vivo and in vitro by real-time PCR or in situ hybridization. In SSc fibroblasts, miR-196a expression was normalized by TGF-β small interfering RNA. miR-196a inhibitor leads to the overexpression of type I collagen in normal fibroblasts, whereas overexpression of the miRNA resulted in the downregulation of type I collagen in SSc fibroblasts. In addition, miR-196a was detectable and quantitative in the serum of SSc patients. Patients with lower serum miR-196a levels had significantly higher ratio of diffuse cutaneous SSc:limited cutaneous SSc, higher modified Rodnan total skin thickness score, and higher prevalence of pitting scars than those without. miR-196a may play some roles in the pathogenesis of SSc. Investigation of the regulatory mechanisms of type I collagen expression by miR-196a may lead to new treatments using miRNA.