METTL3 Regulates ATIC Expression via m6A Methylation: Implications for Methotrexate Action in Rheumatoid Arthritis

Background: Rheumatoid arthritis (RA), an autoimmune condition, causes fibroblast-like synoviocytes (FLSs) growth, joint inflammation, and erosion. Methotrexate (MTX), the principal treatment for this illness, has an unknown mechanism. We investigated the potential regulatory mechanism of MTX in RA-related synovial fibroblasts, focusing on whether MTX exerts therapeutic effects via regulating N6-methyladenosine (m6A) modification mediated by methyltransferase like 3 (METTL3), which affects the expression of the key purine metabolism enzyme 5-Aminoimidazole-4-Carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase (ATIC).Methods: The effects of MTX on RA-FLSs proliferation, migration, invasion, cell cycle distribution and apoptosis were assessed using methylthiazolyldiphenyl-tetrazolium bromide (MTT), Transwell assays and flow cytometry. METTL3 and ATIC protein levels were quantified by Western blotting. Functional rescue experiments were performed using METTL3-overexpressing cells, while the underlying molecular mechanism was investigated by methylated RNA immunoprecipitation (MeRIP) and RNA stability assays.Results: RA-FLSs proliferation, migration, and invasion were significantly higher than those of normal FLSs (p < 0.01). The cells entered S phase, and apoptosis decreased. Meanwhile, METTL3 and ATIC protein expression levels were increased (p < 0.01). MTX suppressed RA-FLS proliferation and migration (p < 0.01), triggered G0/G1 phase arrest and apoptosis (p < 0.001), and decreased METTL3 and ATIC protein levels (p < 0.05). METTL3 overexpression restored ATIC protein expression (p < 0.01) and reversed MTX’s cellular functional control on RA-FLSs (p < 0.01). METTL3 mediated m6A modification of ATIC mRNA, which elevated the m6A enrichment level of the ATIC transcript (p < 0.001) and slowed its mRNA degradation (p < 0.05).Conclusion: MTX reduces m6A enrichment in ATIC mRNA by suppressing METTL3, thereby decreasing ATIC expression. This METTL3-ATIC regulatory axis is associated with altered proliferation and apoptosis of RA-FLSs, suggesting a potential epitranscriptomic mechanism that may contribute to MTX’s therapeutic effects. This study provides preliminary in vitro evidence for a novel molecular pathway underlying MTX activity in RA-related synovial fibroblasts and identifies directions for future mechanistic investigation in rheumatoid arthritis.