M⁶A Demethylase Inhibits Osteogenesis of Dental Follicle Stem Cells via Regulating miR-7974/FKBP15 Pathway

N6-methyladenosine (m(6)A) is the most abundant RNA modification, regulating gene expression in physiological processes. However, its effect on the osteogenic differentiation of dental follicle stem cells (DFSCs) remains unknown. Here, m(6)A demethylases, the fat mass and obesity-associated protein (FTO), and alkB homolog 5 (ALKBH5) were overexpressed in DFSCs, followed by osteogenesis assay and transcriptome sequencing to explore potential mechanisms. The overexpression of FTO or ALKBH5 inhibited the osteogenesis of DFSCs, evidenced by the fact that RUNX2 independently decreased calcium deposition and by the downregulation of the osteogenic genes OCN and OPN. MiRNA profiling revealed that miR-7974 was the top differentially regulated gene, and the overexpression of m(6)A demethylases significantly accelerated miR-7974 degradation in DFSCs. The miR-7974 inhibitor decreased the osteogenesis of DFSCs, and its mimic attenuated the inhibitory effects of FTO overexpression. Bioinformatic prediction and RNA sequencing analysis suggested that FK506-binding protein 15 (FKBP15) was the most likely target downstream of miR-7974. The overexpression of FKBP15 significantly inhibited the osteogenesis of DFSCs via the restriction of actin cytoskeleton organization. This study provided a data resource of differentially expressed miRNA and mRNA after the overexpression of m(6)A demethylases in DFSCs. We unmasked the RUNX2-independent effects of m(6)A demethylase, miR-7974, and FKBP15 on the osteogenesis of DFSCs. Moreover, the FTO/miR-7974/FKBP15 axis and its effects on actin cytoskeleton organization were identified in DFSCs.