Study On The Role And Mechanism Of Alkbh1 In Regulating The Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objectives: DNA N6-methyladenine(N6-mA)demethylase Alkbh1 participates in regulating osteogenic differentiation of human mesenchymal stem cell(MSCs)and vascular calcification.However,the role of Alkbh1 in bone metabolism has not yet been fully elucidated.The purpose this study is to study the role and mechanism of Alkbh1 in regulating the differentiation of bone marrow mesenchymal stem cells(BMSCs)and bone metabolism.Materials and Methods: The expression profile of Alkbh1 was analyzed by the Genotype Tissue Expression(GTEx)in different human tissues.Western blot,qRT-PCR and immunofluorescent staining were used to evaluate the expression of Alkbh1 in mouse BMSCs.BMSCs specific Alkbh1 knockout mice were used to investigate the role of Alkbh1 in bone metabolism.Micro-CT,HE staining,immunohistochemical staining,Trap staining and calcein double labelling assay were used to evaluate bone phenotypes of mice.Alizarin Red staining(ARS),Alkaline phosphatase(ALP)staining,oil red O staining and qRT-PCR were used to evaluate the osteogenic or adipogenic differentiation of BMSCs.Dot blotting was used to detect the level of N6-mA in genomic DNA.Gene Expression Omnibus(GEO)database and Chromatin immunoprecipitation(ChIP)assays were used to identify critical targets of Alkbh1.Alkbh1 adeno-associated virus were used to overexpress Alkbh1 in BMSCs of aged mice.Results: Alkbh1 expression in BMSCs declined during aging.Knockout of Alkbh1 promoted adipogenic differentiation of BMSCs while inhibited osteogenic differentiation.BMSCs specific Alkbh1 knockout mice exhibited reduced bone mass and increased marrow adiposity.Mechanistically,we identified Optn as the downstream target through which Alkbh1-mediated DNA N6-mA modification regulated BMSCs fate.Overexpression of Alkbh1 attenuated bone loss and marrow fat accumulation in aged mice.Conclusions: Our findings demonstrated that Alkbh1 regulated BMSCs fate and bone-fat balance during skeletal aging and provided a potential new target for the treatment of osteoporosis.