Background We recently found that miR-145 plays anti-fibrotic effects in SSc fibroblasts via the regulation of the TGF-β pathway in a multi-step fashion.
Objectives To elucidate the mechanisms regulating the expression of miR-145 in SSc fibroblasts.
Methods Basal miR-145 and pri-miR-145 expression was analysed in 12 SSc and 6 healthy control (HC) dermis and fibroblasts by Real-time PCR. MiR-145 expression was further analysed in SSc and HC fibroblasts exposed to hypoxia, stimulated with pro-fibrotic cytokines (TGF-β, PDGF-B, VEGF-A), and treated with either a Smad3 inhibitor (SIS3) or a TGF- βRII neutralizing antibody. Finally, miR-145 expression was analysed in peripheral blood mononuclear cells (PBMCs) of 5 SSc patients and 5 HC.
Results MiR-145 was down-regulated in both SSc dermis and fibroblasts by 2.2 and 2.0 fold respectively (p<0.001) as compared to HC. Similar results were obtained for the non-functional primary transcript, pri-miR-145, suggesting a regulation on the transcriptional level rather than post-transcriptional modifications. No significant difference in the expression of miR-145 was found between SSc fibroblasts exposed to hypoxia or pro-fibrotic cytokines TGF-β, PDGF-B, VEGF-A at conventional doses compared to controls. However, treatment of SSc fibroblasts with both SIS3 and TGF-βRII neutralizing antibodies increased miR-145 levels by 2.4 and 1.8 fold, respectively. This prompted us to investigate the expression of miR-145 in HC fibroblasts treated with TGF-β at 5 ng/ml, finding a 1.3 fold down-regulation, suggesting a possible regulatory loop between miR-145 and the TGF-β pathway. In addition, down-regulation of miR-145 in SSc was mediated by epigenetic modifications, as inhibition of DNA methyltransferases by 5aza-C increased miR-145 levels by 2.5 fold in SSc fibroblasts (p<0.01). Finally, to clarify whether miR-145 down-regulation in SSc fibroblasts was a cell-specific finding, we investigated miR-145 expression in SSc PBMCs and found no difference with respect to HC (p>0.05).
Conclusions We previously showed that the down-regulation of miR-145 in SSc has strong pro-fibrotic effects in SSc fibroblasts via direct post-transcriptional induction of TGF-β signalling (TGFBR2 and SMAD3) and CTGF, directly interacting with TGFBR2 3’UTR. Our new data suggest that miR-145 down-regulation in SSc is rather specific for skin fibroblasts. In addition, miR-145 expression in SSc fibroblasts appears modulated by epigenetic mechanisms. Most interestingly, there is a feedback regulation between TGF-β and miR-145 which self-enhances the pathologic pathway. Taken together, these data further support the role for miR-145 in the regulation of the TGF-β pathway in SSc, and make the restoration of miR-145 levels an appropriate option for the treatment of the disease.
Disclosure of Interest S. Vettori Grant/Research support from: EULAR ODP, M. Brock: None Declared, N. Iwamoto: None Declared, B. Maurer: None Declared, A. Jungel: None Declared, R. Gay: None Declared, M. Calcagni: None Declared, G. Valentini: None Declared, J. Distler: None Declared, S. Gay: None Declared, O. Distler Grant/Research support from: EULAR ODP, Actelion, Pfizer, Ergonex, Sanofi, Consultant for: Actelion, Pfizer, Ergonex, BMS, Sanofi-Aventis, United BioSource Corporation, medac, 4D Science, Boehringer-Ingelheim, Active Biotech, Roche, Speakers Bureau: Actelion, Pfizer, Encysive, Ergonex