Background: N6-methyladenosine (m6A) is a highly enriched modification found in circular RNAs (CircRNAs); however, the ability and mechanism of CircRNAs to encode for m6A function in rheumatoid arthritis (RA) remain poorly understood.
Methods: We utilized an epitranscriptomic microarray to measure levels and quantities of m6A methylated CircRNAs in synovial tissues of patients with RA and osteoarthritis (OA). We then utilized methylated RNA immunoprecipitation- and MazF-quantitative PCR to identify and validate differentially m6A-methylated RNAs between the groups, conducted a functional enrichment analysis, and selected protein-protein interaction hub genes. Lastly, we predicted and validated the CircRNA/miRNA/mRNA interaction networks.
Results: We detected 4,845 CircRNAs containing m6A in our samples, with 53 CircRNAs upregulated, and 139 CircRNAs downregulated compared to human OA synovial tissue (|fold change| ≥ 1.2 and p ≤ 0.05). The differentially m6A-modified CircRNAs were associated with the interleukin-6-mediated signaling pathway, with an increase in relative m6A-methylated levels of hsa_circ_0007259 in human RA, a significant decrease in hsa_miR-21-5p, and an increase in signal transducer and activator of transcription 3（STAT3）. The Luciferase Reporter Gene assay verified the binding of hsa_circ_0007259 to hsa_miR-21-5p and the subsequent binding of hsa_miR-21-5p to STAT3.
Conclusion: We showed a notable increase in the relative m6A-methylated levels of hsa_circ_0007259 in human RA, indicating a potential role of hypermethylated hsa_circ_0007259 in RA pathogenesis. This may provide valuable insight into the mechanism of RA and the possibility of utilizing hsa_circ_0007259 as a valuable biomarker.
Keywords: Circular RNA; N6-methyladenosine; Osteoarthritis; Rheumatoid arthritis; m6A methylation expression.
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