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OP0018 Microrna-145 down-regulation in systemic sclerosis is limited to skin fibroblasts and influenced by epigenetic modifications and negative feedback loop by TGF-β
  1. S. Vettori1,2,
  2. M. Brock2,
  3. N. Iwamoto2,
  4. B. Maurer2,
  5. A. Jungel2,
  6. R.E. Gay2,
  7. M. Calcagni3,
  8. G. Valentini4,
  9. J.J. Distler5,
  10. S. Gay2,
  11. O. Distler2
  1. 1Department of Internal Medicine, Cardiology and Immunology, Federico I I University of Naples, Naples, Italy
  2. 2Center of Experimental Rheumatology
  3. 3Division of Plastic Surgery and Hand Surgery, Zurich University Hospital, Zurich, Switzerland
  4. 4Rheumatology Unit, Second University of Naples, Naples, Italy
  5. 5Department of Internal Medicine 3, Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany

Abstract

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

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