Abstract

N 6 -methyladenosine (m 6 A) is the most abundant posttranscriptional methylation modification that occurs in mRNA and modulates the fine-tuning of various biological processes in mammalian development and human diseases. In this study we investigated the role of m 6 A modification in the osteogenesis of mesenchymal stem cells (MSCs), and the possible mechanisms by which m 6 A modification regulated the processes of osteoporosis and bone necrosis. We performed systematic analysis of the differential gene signatures in patients with osteoporosis and bone necrosis and conducted m 6 A-RNA immunoprecipitation (m 6 A-RIP) sequencing to identify the potential regulatory genes involved in osteogenesis. We showed that fat mass and obesity (FTO), a primary m 6 A demethylase, was significantly downregulated in patients with osteoporosis and osteonecrosis. During the differentiation of human MSCs into osteoblasts, FTO was markedly upregulated. Both depletion of FTO and application of the FTO inhibitor FB23 or FB23-2 impaired osteogenic differentiation of human MSCs. Knockout of FTO in mice resulted in decreased bone mineral density and impaired bone formation. PPARG, a biomarker for osteoporosis, was identified as a critical downstream target of FTO. We further revealed that FTO mediated m 6 A demethylation in the 3’UTR of PPARG mRNA, and reduced PPARG mRNA stability in an YTHDF1-dependent manner. Overexpression of PPARG alleviated FTO-mediated osteogenic differentiation of MSCs, whereas knockdown of PPARG promoted FTO-induced expression of the osteoblast biomarkers ALPL and OPN during osteogenic differentiation. Taken together, this study demonstrates the functional significance of the FTO-PPARG axis in promoting the osteogenesis of human MSCs and sheds light on the role of m 6 A modification in mediating osteoporosis and osteonecrosis.