Regulation Of M~6A Methyltransferase METTL3 On Osteogenic Differentiation Of Human Adipose Stem Cells

Objective:N6-methyladenosine(m~6A)modification is the most common m RNA epigenetic modification in all higher eukaryotes.It has dynamic reversibility and regulates many complex cellular processes,such as RNA processing,transportation,localization,translation,and degradation.Methyltransferase-like 3(METTL3)is the most active enzyme in the m~6A methylation modification process.In bone biology,METTL3-mediated m~6A alteration is essential for bone growth and conditions,including osteoporosis,osteoarthritis,and osteosarcoma that affect the bone.Adipose-derived stem cells(ADSCs)are mesenchymal stem cells with multiple differentiation potentials and the ability to self-renew.It comes from a variety of sources and is simple to obtain.It’s a fantastic seed cell for bone tissue engineering.METTL3 and m~6A research currently focuses on bone marrow mesenchymal stem cells.This study aims to investigate the relationship between METTL3 and osteogenic differentiation of hADSCs,as well as to provide a new perspective on the molecular mechanism of this process.Methods:1.Primary hADSCs were extracted from human adipose tissue,and surface markers of hADSCs were identified using flow cytometry.2.Western blot and RT-PCR experiments were performed on hADSCs of osteogenic induction and differentiation for 0,3,7,and 14 days to detect the expression changes of METTL3 at different times of osteogenic induction and differentiation3.siRNA was transfected into hADSCs to reduce METTL3 expression,and osteogenic differentiation was observed at 0,3,7,and 14 days after transfection.ALP staining and Alizarin red staining were carried out,and OCP Western blot experiments were performed to detect the osteogenic differentiation levels of hADSCs after siRNA transfection.Results:1.Primary hADSCs can differentiate into multiple potentials,and flow cytometry results show that CD45 is negative on the surface of hADSCs,while CD73,CD90,and CD105 are positive.;2.The expression level of METTL3 increased during the osteogenic differentiation of hADSCs,and silencing METTL3 decreased the expression levels of RUNX2 and OCN.Alizarin red staining and ALP staining suggested that silencing METTL3weakened the osteogenic differentiation ability of hADSCs.Conclusions:The expression level of METTL3 gradually increased during the osteogenic differentiation of hADSCs,and silencing METTL3 inhibited the osteogenic differentiation of hADSCs.