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AB0837 Circulating micrornas in systemic sclerosis patients reflect microrna profile of cultured scleroderma fibroblasts and correlate with distinct disease features
  1. S. Vettori1,2,
  2. B. Maurer2,
  3. N. Iwamoto2,
  4. M. Filkova2,
  5. G. Cuomo3,
  6. R.E. Gay2,
  7. G. Valentini3,
  8. S. Gay2,
  9. O. Distler2
  1. 1Department of Internal Medicine, Cardiology and Immunology, Federico I I University of Naples, Naples, Italy
  2. 2Center of Experimental Rheumatology, University of Zurich, Zurich, Switzerland
  3. 3Rheumatology Unit, Second University of Naples, Naples, Italy

Abstract

Background MicroRNAs (miRs) play a role in many pathophysiologic processes, and have been proposed as tissue-based biomarkers, which are released and stably detectable in body fluids.

Objectives To analyse serum expression of miRs which are down-regulated in cultured Systemic Sclerosis (SSc) skin fibroblasts and look for correlations with SSc patient’s features.

Methods Pooled RNA from 3 SSc and 3 healthy control (HC) fibroblasts was used to screen the expression of 377 miRs by low-density array, and RNA from 12 individual SSc and 6 HC fibroblasts was used to confirm the results by Real time PCR. Serum was obtained from 40 SSc patients and 40 HCs. RNA was isolated by triple phenol-chloroform extraction, from 1 ml serum after adding a denaturing reagent and 62.5 fmol of synthetic cel-miR-39 to allow for normalization of total RNA among samples. A fixed volume of 1.67 μl RNA was used in each specific miR reverse transcription reaction. MiR expression was analysed by TaqMan Real-time PCR and dCt method using cel-miR-39 as control miR.

Results Nine out of the 26 miRs found to be down-regulated in SSc fibroblasts by low-density array were confirmed by Real-time PCR (miR-17-5p, miR-20a, miR-21, miR-24, miR-29a, miR-99a, miR-145, miR-186, and miR-193b; all p<0.05) and were investigated in serum samples. MiR-193b and miR-99a showed very low expression levels in HC sera and were not detectable in 20/40 and 12/40 SSc samples. MiR-17-5p was also not detectable in 12/40 SSc samples. Therefore, these miRs were excluded from further analysis. MiR-20a (p=0.003), miR-21 (p=0.002), miR-29a (p<0.0001), miR-145 (p=0.0001), and miR-186 (p=0.02) were down-regulated in serum from SSc patients as compared to HCs, whereas miR-24 showed no difference between the two groups. In SSc patients, levels of miR-29a and miR-186 were lower in patients with dcSSc subset (p=0.004; p=0.03), lung fibrosis (p=0.01; p=0.008), and FVC <80% (both p=0.001) on univariate analysis. In addition, both miRs negatively correlated with the modified Rodnan Skin Score (mRSS) (p=0.0002; p=0.006). MiR-21 levels were lower in patients with lcSSc subset (p=0.03), ACA positivity (0.002), digital ulcers (0.0004), and positively correlated with DLCO (p=0.02). MiR-20a levels were lower in patients with Scl-70 positivity (p=0.001) and free from digital ulcers (0.002), and negatively correlated with mRSS (p=0.02). MiR-145 levels did not show differences between disease subsets and patterns of organ involvement, but interestingly were affected by previous intake of cyclophosphamide (p=0.002). The association of low serum levels of miR-29a and miR-186 with FVC <80%, and the positive correlation of miR-20a with Scl-70 antibodies and the negative correlation with digital ulcers was confirmed on multivariate analysis.

Conclusions Most miRs down-regulated in cultured SSc fibroblasts are detectable in SSc serum samples and display a similar expression profile. Different miRs were associated with distinct SSc features at univariate analysis. Of note, miR-29a and miR-186 were independently associated to FVC <80% at multivariate analysis, and might be considered as candidate biomarkers for fibrotic lung involvement in SSc patients.

Disclosure of Interest S. Vettori Grant/Research support from: EULAR ODP, B. Maurer: None Declared, N. Iwamoto: None Declared, M. Filkova: None Declared, G. Cuomo: None Declared, R. Gay: None Declared, G. Valentini: 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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