FRI0359 Methyl Donors Change the Expression Profile of Mir-203 and Mir-29C in Rheumatoid Arthritis Synovial Fibroblasts
Authors: N. Gaur, E. Karouzakis, A. Jüngel, C. Kolling, M. Beat, R.E. Gay, S. Gay, M. Neidhart
Journal: Annals of the Rheumatic Diseases (2014)

<h3>Background</h3> 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. <h3>Objectives</h3> 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. <h3>Methods</h3> 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. <h3>Results</h3> 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%. <h3>Conclusions</h3> 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. <h3>Acknowledgements</h3> Institute for Arthritis Research Epalinges (IAR) and IMI BTCure <h3>Disclosure of Interest</h3> 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 <h3>DOI</h3> 10.1136/annrheumdis-2014-eular.4436