Genome-wide profiling of m6A modification and its role in the transcriptome of sequelae of pelvic inflammatory disease

Sequelae of pelvic inflammatory disease (SPID), such as chronic pelvic pain and infertility, are major long-term complications that significantly harm women's health. N6-methyladenosine (m6A), the most common internal mRNA modification in eukaryotes, is recognized as a key regulator of gene expression in inflammatory and immune processes, yet its role in SPID pathogenesis remains unclear. This study aimed to investigate the relationships between SPID and m6A methylation of related genes and mRNAs, explore the role of m6A-related mRNAs in SPID, and provide a reference for its clinical application. The discovery of m6A-methylated mRNAs may offer a new perspective for developing therapeutic drugs for SPID. High-throughput sequencing combined with methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing was used to analyze differential m6A methylation and mRNA expression profiles in the peripheral blood of 5 SPID patients and 5 healthy controls. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to explore differentially methylated and expressed mRNAs. Methylated RNA Immunoprecipitation (MeRIP) - Quantitative Real-time Polymerase Chain Reaction (qPCR) and Reverse Transcription (RT)-qPCR were subsequently used to verify the top 3 differentially expressed mRNAs in terms of m6A methylation and mRNA expression levels. Among the significantly differentially methylated genes, 65 were hypermethylated and 140 were demethylated. GO analysis indicated that these genes were primarily involved in mRNA processing, RNA splicing, and neutrophil-mediated immune response. KEGG analysis revealed associations with pathways such as leukocyte transendothelial migration, natural killer cell-mediated cytotoxicity, and the mTOR signaling pathway. RNA-seq results showed 74 upregulated and 116 downregulated genes. GO and KEGG analyses of differentially expressed genes revealed enrichment in the negative regulation of immune system processes and angiogenesis, as well as antigen processing and presentation. Integration of MeRIP-seq and RNA-seq identified 6 genes with significant differences in both m6A modification and mRNA expression, 3 of which were verified by methylated RNA immunoprecipitation (MeRIP)-qPCR and RT-qPCR, with consistent trends with sequencing results. This study established the m6A transcriptional profile in the peripheral blood of SPID patients, revealed potential relationships between mRNA methylation and SPID-related genes, and suggested that m6A modification may be related to the occurrence and progression of SPID.