Background Rheumatoid arthritis synovial fibroblasts (RASF) show global DNA hypomethylation mirrored in their intrinsically activated and aggressive phenotype. Methyl donors, such as L-methionine (LM) and betaine (BT), as diet supplements feed into the methionine cycle donating methyl groups for transmethylation reactions, including DNA methylation. Methyl donors represent a novel nutriepigenomic treatment strategy to reverse hypomethylation not only locally in RA synovium but also systemically in peripheral T lymphocytes of RA patients.

Objectives To investigate the effects of methyl donors on the expression of enzymes involved in DNA methylation pathways and of their regulatory microRNAs (miRs).

Methods RNA was extracted from RASF (n=8-11) treated with 0, 5 or 50mM LM or BT for 14 days. The expression of miRs predicted or validated to target epigenetic enzymes involved in DNA methylation, methionine and polyamine cycles was measured by qPCR. RASF were transfected with pre- and antago-miR-29c or Let-7f to confirm their mRNA targets. Homocysteine levels in the culture supernatants of RASF were measured by ELISA.

Results BT and LM significantly altered the miR profile in RASF. miR-29c levels were significantly up regulated by 50mM BT treatment (x-fold ± SD: 1.73±0.76, p=0.03), while Let-7f levels were significantly increased by both 50mM LM and 50mM BT (2.04±0.61, p<0.001; and 1.93±0.93, p=0.02, respectively). LM increased miR-196a and BT elevated miR-9 and miR-203 levels. Additionally, several enzymes of transmethlyation cycles were significantly altered by methyl donor treatment. mRNA levels of DNA methyltransferases (DNMT) 1 (0.56±0.13; p<0.0001), DNMT3A (0.61±0.17; p<0.0001) and DNMT3B (0.72±0.22; p=0.004) were repressed upon 50mM BT treatment. LM up regulated, and BT down regulated the mRNA levels of adenosylhomocysteinase (AHCY) and adenosyl methionine decarboxylase 1 (AMD1). Betaine homocysteine methyl transferase (BHMT) and spermidine/spermine N1-acetyltransferase (SSAT1) mRNAs were not changed. The protein levels of DNMT1, AMD1, SSAT1, and BHMT were significantly elevated by BT, and even more by LM, suggesting a substrate-dependent stabilization of these enzymes. Transfection experiments showed that in RASF miR-29c, but not let-7f, directly targets DNMT3A. Most importantly, antago-miR-29c normalized the levels of DNMT3A mRNA that were repressed by LM and BT. LM, but not BT significantly increased secretion of homocysteine from RASF.

Conclusions Investigating the effects of methyl donors as nutriepigenomic agents is important to understand their therapeutic potential in RA. Our study suggests that alterations in cellular microRNA profiles, in particular the up regulation of miR-29c, which targets DNMT3A, may limit the efficiency of methyl donors as DNA re-methylating agents. MiR-29c is known to regulate many cellular processes and here we show that, repressing miR-29c during betaine supplementation may enhance its DNA remethylation efficiency. In contrast to methionine, betaine does not increase the levels of homocysteine - a known cardiovascular risk factor, which makes betaine a superior candidate for therapeutic use.

Disclosure of Interest N. Gaur: None declared, E. Karouzakis: None declared, A. Jungel: None declared, M. Frank-Bertoncelj: None declared, E. Bagdonas: None declared, C. Kolling: None declared, B. A. Michel: None declared, R. E. Gay: None declared, S. Gay Grant/research support from: IAR, M. Neidhart Grant/research support from: Baugarten Stiftung