Integrated single-cell and transcriptomic analyses uncover m6A-related molecular alterations in endometriosis

Endometriosis (EM) is a common gynecological disease. Though m6A RNA methylation has emerged as a key regulator in various physiological and pathological processes, its cell-type-specific function in EM remains unclear. We obtain batch and single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database. The “Seurat” package was used to annotate and label cell types in scRNA-seq data. After identifying m6A-associated modules with weighted gene co-expression network analysis (WGCNA), core biomarkers for EM were filtered and selected by conducting least absolute shrinkage and selection operator (LASSO), support vector machine–recursive feature elimination (SVM–RFE), and eXtreme Gradient Boosting (XGBoost). Subsequently, functional enrichment analysis was performed with the "clusterProfiler" package. Immune infiltration and pathway activity were assessed by single-sample gene set enrichment analysis (ssGSEA). Drug prediction was performed using DSigDB in Enrichr tool, and molecular docking was conducted with AutoDock Vina. Finally, quantitative real-time PCR (qRT-PCR) and Western blot were performed to detect gene expression in vitro, and cell migration and invasion were assessed by carrying out wound healing and Transwell assays. The scRNA-seq analysis revealed altered cellular composition in EM, characterized by increased proportions of epithelial and NK/T cells. m6A regulatory activity was significantly elevated in EM samples, particularly in immune cells, and was associated with neutrophil activation and RNA splicing pathways. Integrated analysis of WGCNA and machine learning identified EPCAM, DST, HSPH1, and NAP1L1 as core m6A-related genes. These four genes were correlated with specific immune cell subsets, including immature dendritic cells and effector memory CD4 + T cells, and were enriched in cell cycle-related oncogenic pathways. Drug prediction revealed that dronabinol and minocycline might be potential therapeutics for EM, with molecular docking confirming favorable binding affinities. Functionally, EPCAM was downregulated in endometriotic 12Z cells, and its overexpression significantly suppressed cell migration. This study revealed that m6A-related dysregulation contributed to EM pathogenesis through dysregulated immune responses and affected cell migration. The identified gene signature and candidate drugs provide novel insights for potential therapeutic strategies.