The critical role of m(6)A methylation in the pathogenesis of Graves' ophthalmopathy

PURPOSE: To investigate the role of N6-methyladenosine (m(6)A) RNA modification in the pathogenesis of Graves' ophthalmopathy (GO). METHODS: Surgically excised extraocular muscles from 7 patients with GO and 5 subjects without GO were used. The global m(6)A levels in the specimens were determined using an m(6)A RNA methylation quantification kit. RNA sequencing (RNA-seq) was used to analyze the molecules involved in the regulation of m(6)A RNA methylation and the differential expression of mRNAs between the two groups (4 eyes, respectively). The expression of m(6)A RNA modification genes was evaluated by real-time PCR. The functional implications of the gene alterations between the GO and control specimens were determined by Gene Ontology analysis. RESULTS: The m(6)A level was significantly increased in the specimens of GO patients compared to the control specimens (P < 0.05). The expression of m(6)A methylation regulators, such as WT1 associated protein (WTAP), alkylation repair homolog protein 5 (ALKBH5), E74 like ETS transcription factor 3 (ELF3), YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), YTHDF3 and YTH domain containing 2 (YTHDC2), was significantly upregulated (P < 0.05). Gene Ontology enrichment analysis showed that the most highly upregulated genes and biological pathways were related to the immune response and inflammatory processes such as lymphocyte activation, leukocyte differentiation, cytokine production and cytokine-mediated signaling pathways. CONCLUSIONS: Our results suggest that m(6)A methylation may play a critical role in the pathogenesis of GO and that targeting genes that regulate m(6)A methylation may provide a new therapeutic approach for GO.