N6-methyladenosine (m6A) is the most frequent internal modification in eukaryotic mRNAs and is closely related to the occurrence and development of many diseases, especially
tumors. However, the relationship between
m6A methylation and
rheumatoid arthritis (RA) is still a mystery.
METHODS: Two high-throughput sequencing methods, namely,
m6A modified
RNA immunoprecipitation sequence (m6A-seq) and RNA sequence (
RNA-seq) were performed to identify the differentially expressed
m6A methylation in human
rheumatoid arthritis fibroblast-like synoviocytes cell line MH7A after stimulation with TNF-α. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to obtain enriched GO terms and significant KEGG pathways. Then, four candidate genes, Wilms tumor 1-associating
protein (WTAP), receptor-interacting
serine/threonine protein kinase 2 (RIPK2),
Janus kinase 3 (JAK3) and
tumor necrosis factor receptor SF10A (TNFRSF10A) were selected to further validate the
m6A methylation,
mRNA and
protein expression levels in MH7A cells and synovial tissues of
adjuvant arthritis (AA) rats by RT-qPCR and Western blot.
RESULTS: Using m6A-seq, we identified a total of 206 genes with differentially expressed
m6A methylation, of which 118 were significantly upregulated and 88 genes were significantly downregulated. Likewise, 1207 differentially
mRNA expressed mRNAs were obtained by
RNA-seq, of which 793 were upregulated and 414 downregulated. Further joint analysis showed that the
m6A methylation and
mRNA expression levels of 88 genes changed significantly, of which 30 genes displayed increased
m6A methylation and decreased
mRNA expression, 57 genes displayed decreased
m6A methylation and increased
mRNA expression increased, and 1 gene displayed increased
m6A methylation and increased
mRNA expression. GO and KEGG analyses indicated that these unique genes were mainly enriched in
inflammation-related pathways, cell proliferation and apoptosis. In addition, the validations of WTAP, RIPK2, JAK3 and TNFRSF10A were in accordance with the
m6A and
RNA sequencing results.
CONCLUSION: This study established the transcriptional map of
m6A in MH7A cells and revealed the potential relationship between RNA methylation modification and RA related genes. The results suggested that
m6A modification was associated with the occurrence and course of RA to some extent.