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FRI0359 Methyl Donors Change the Expression Profile of Mir-203 and Mir-29C in Rheumatoid Arthritis Synovial Fibroblasts
  1. N. Gaur1,
  2. E. Karouzakis2,
  3. A. Jüngel1,
  4. C. Kolling3,
  5. M. Beat2,
  6. R.E. Gay2,
  7. S. Gay2,
  8. M. Neidhart2
  1. 1Center of Experimental Rheumatology
  2. 2University Hospital Zurich
  3. 3Schulthess Clinic, Zurich, Switzerland

Abstract

Background Rheumatoid arthritis synovial fibroblasts (RASF) display a global DNA hypomethylation. Many microRNAs (miRs) are regulated by promoter methylation. Previously, it has been described that the basal expression of miR-203 is higher in RASF compared to osteoarthritis synovial fibroblasts (OASF) or healthy fibroblasts and its expression increased upon treatment with the hypomethylating drug 5-azacytidine. On the other hand, the expression of miR-29c is down-regulated in many cancers, what has been paradoxically associated with DNA hypomethylation. DNA methylation can be restored by methyl donors such as L-Methionine and Betaine, which in turn can regulate the expression of miRs.

Objectives To investigate whether the expression of these methylation-sensitive miRs (miR-203 and miR-29c) can be modulated by methyl donors, such as L-Methionine and Betaine.

Methods RASF (n=3) were supplemented with 0.5mM, 5mM and 50mM L-Methionine or Betaine for 14 days. Flow cytometric analysis was performed using Annexin V/Propidium iodide staining to exclude any cytotoxic or apoptotic effects of these supplements. RNA was isolated from RASF. The expression of miR-203 and miR-29c was determined by Taqman Real-time PCR with normalization to RNU6B.

Results Flow cytometry did not reveal any cytotoxic or apoptotic effects of the added supplements on RASF. After the treatment of RASF with 50mM L-Methionine or 50mM Betain, the expression of miR-203 was reduced by 50±5% and 62±14%, respectively. Therefore both, L-Methionine and Betaine, achieved normalization of the increased levels of miR-203 in RASF, as compared with OASF. These data are in accordance with our previous observations of the increased levels of miR-203 in cells, which were DNA hypomethylated with 5-azacytidine. We could also show that the basal expression of miR-29c is lower in RASF as compared to OASF (by 18±9%). Interestingly, the expression of miR-29c was restored to normal levels in RASF treated with 50mM L-Methionine, i.e. up regulated by 18±2%; 50mM Betaine, however, down regulated the levels of miR-29c in RASF by 20±13%.

Conclusions Our results confirmed the methylation-dependency of miR-203 and miR-29c. The effect of methyl donors on the methylation of miR-203 could be explained by an increased methylation of the miR-203 promoter. Interestingly, miR-29c reacted in an opposite way upon the treatment with L-Methionine, suggesting an indirect methylation-sensitive regulation mechanism. We conclude that both miR-203 and miR-29c could be useful epigenetic biomarkers during the treatment of RA with methyl donors.

Acknowledgements Institute for Arthritis Research Epalinges (IAR) and IMI BTCure

Disclosure of Interest N. Gaur: None declared, E. Karouzakis Grant/research support: Institute for Arthritis Research Epalinges (IAR) and IMI BTCure, A. Jüngel Grant/research support: Institute for Arthritis Research Epalinges (IAR) and IMI BTCure, C. Kolling: None declared, M. Beat: None declared, R. Gay Grant/research support: Institute for Arthritis Research Epalinges (IAR) and IMI BTCure, S. Gay Grant/research support: Institute for Arthritis Research Epalinges (IAR) and IMI BTCure, M. Neidhart Grant/research support: Institute for Arthritis Research Epalinges (IAR) and IMI BTCure

DOI 10.1136/annrheumdis-2014-eular.4436

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