A novel mechanism of FTO modulating the progression of endometriosis through mediating the m6A methylation of GEF-H1 in a YTHDF1-dependent manner

Abstract Background Endometriosis (EMs) is a condition characterized by the growth of endometrial tissue outside the uterine cavity. Although this condition is benign, it has cancer-like features. N6-methyladenosine (m6A) is a common RNA modification involved in diverse biological processes, but its role in EMs remains unclear. Methods A human endometrial stromal cell line (HESCs), primary eutopic endometrial stromal cells (Eu-ESCs), primary ectopic endometrial stromal cells (Ec-ESCs), and clinical samples were used in this study. A colorimetric assay was used to measure methylation levels in clinical and mouse EMs samples. Functional assays (CCK-8, EdU, Transwell, and wound healing) were used to evaluate phenotypic changes. m6A immunoprecipitation sequencing (MeRIP-seq) identified downstream targets. Mechanistic studies were conducted via qRT‒PCR, Western blot, RNA immunoprecipitation (RIP), dual-luciferase reporter, and RNA stability assays. Results We detected aberrantly low levels of m6A within endometriotic lesions, which was attributed to increased expression of the m6A eraser fat mass and obesity-associated protein (FTO). Notably, estrogen and inflammatory factors, which are recognized as pathogenic agents in EMs amplify FTO expression while suppressing m6A levels. In vitro experiments demonstrated that overexpression of FTO in endometrial stromal cells leads to a reduction in m6A levels and concomitantly promotes their proliferation, migration, and invasion. Furthermore, both genetic deletion of Fto and chemical inhibition of FTO impeded the growth of ectopic endometrial lesions in vivo. By utilizing m6A-seq, we identified GEF-H1 (a Rho guanine nucleotide exchange factor) as a pivotal downstream target of FTO. Specifically, diminished m6A methylation at a certain site within the 3'UTR of GEF-H1 promotes its expression in a YTH N6-methyladenosine RNA-binding protein F1 (YTHDF1)-dependent manner, thereby activating the RhoA pathway. Subsequent experiments revealed that GEF-H1 mediates the effects of FTO in promoting migration and invasion. Conclusions This study revealed that FTO decreases the m6A level of GEF-H1 , thereby increasing its stability, which in turn activates the GEF-H1-RhoA pathway to promote the migration and invasion of endometrial stromal cells, thereby inducing EMs. Our findings suggest potential therapeutic avenues for targeting FTO to alleviate EMs progression.