N6-methyladenosine Demethylase FTO Induces the Dysfunctions of Ovarian Granulosa Cells by Upregulating Flotillin 2

Polycystic ovarian syndrome (PCOS) is often accompanied by overweight/obesity and insulin resistance. The dysfunctions of ovarian granulosa cells (GCs) are closely linked with the pathogenesis of PCOS. Fat mass and obesity-associated gene (FTO), an N6-methyladenosine (m6A) demethylase, has been reported to be implicated in the risks and insulin resistance of PCOS. However, the roles of FTO in the development of GCs along with its m6A-related regulatory mechanisms are poorly defined. Cell proliferative ability was detected by MTT assay. Cell apoptotic rate was measured via flow cytometry. Insulin resistance was assessed by GLUT4 transport potential. The mRNA and protein levels of FTO and flotillin 2 (FLOT2) were determined by RT-qPCR and western blot assays, respectively. FLOT2 was screened out to be a potential FTO target through differential expression analysis for the GSE95728 dataset and target prediction analysis by POSTAR2 and STARBASE databases. The interaction between FTO and FLOT2 was analyzed by RNA immunoprecipitation (RIP) assay. The effect of FTO upregulation on FLOT2 m6A level was measured by methylated RIP (meRIP) assay. FLOT2 mRNA stability was examined by actinomycin D assay. FTO overexpression facilitated cell proliferation, hindered cell apoptosis, and induced insulin resistance in GCs. FTO promoted FLOT2 expression by reducing m6A level on FLOT2 mRNA and increasing FLOT2 mRNA stability. FLOT2 loss weakened the effects of FTO overexpression on cell proliferation/apoptosis and insulin resistance in GCs. FTO induced the dysfunctions of GCs by upregulating FLOT2, suggesting that FTO/FLOT2 might play a role in the pathophysiology of PCOS.