Effects of Dishevelled Protein DNA Methylation in Wnt Pathway on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Osteoporosis Patients
Objective:To investigate the effects of dishevelled protein(DVL)DNA methylation in Wnt pathway on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in osteoporosis(OP)patients.Methods:BMSCs from OP patients were isolated and cultured,BMSCs were cultured for osteogenic induction for 0,7,14 and 21 days,the morphological changes of BMSCs were observed,alkaline phosphatase(ALP)staining and activity were detected,alizarin red staining and calcium nodule formation were detected,the expression of distal deletion homeobox 5(Dlx5),core binding protein factor 2(Runx2),osteoblast-specific transcription fac-tor(OSX)and type Ⅰ collagen(CollaI)were detected by fluorescence quantitative polymerase chain reaction(RT-PCR)and immunoblot-ting.The expression of DVL1,Wnt,glycogen synthase kinase 3(GSK3),β-eatenin(β-catenin)and the level of DVL DNA methylation were detected.The methyltransferase inhibitor 5-Aza was added to the osteogenic induction medium,BMSCs were divided into control group(Control group),methyltransferase inhibitor group(5-Aza group),methyltransferase inhibitor+si-NC group(5-Aza+si-NC group)and methyltransferase inhibitor+si-Wnt group(5-Aza+si-Wnt group),ALP activity,alizarin red staining and calcium nodule formation were measured in turn.The levels of Dlx5,Runx2,OSX and Colla 1 were detected by RT-PCR,the protein expression levels of Dlx5,Runx2,OSX,Colla I,DVL1,Wnt,GSK3 and β-catenin were detected by immunoblotting,and DVL DNA methylation level was detected.Results:BMSCs had strong ALP activity and mineralized nodule formation ability after osteogenic induction,with the increase of culture time,the ALP activity and mineralized nodule formation ability of BMSCs cells increased,the mRNA levels of Dlx5,Runx2,OSX and Colla I and the expression of Wnt,GSK3,β-catenin and DVL1 increased,and DVL DNA methylation level decreased(P＜0.05).Com-pared with Control group,ALP staining was deepened,activity was enhanced,and calcium nodule formation was increased in 5-Aza group(P＜0.05),the mRNA and protein expressions of Dlx5,Runx2,OSX,CollaI,Wnt,GSK3,β-catenin and DVL1 were increased,and DVL DNA methylation level was decreased(P＜0.05).Compared with 5-Aza+si-NC group,ALP staining was lighter,ALP activity was decreased,calcium nodule formation was decreased in 5-Aza+si-Wnt group(P＜0.05),Dlx5,Runx2,OSX,CollaI mRNA and protein ex-pression,Wnt,GSK3,β-catenin,DVL1 expression were decreased,and DVL DNA methylation level was increased(P＜0.05).Conclu-sion:DVL DNA methylation can inhibit the osteogenic differentiation of BMSCs in OP patients by inhibiting Wnt/β-catenin signaling pathway.
Wnt pathwayDishevelled DNA methylationOsteoporosisBone marrow mesenchymal stem cellsOsteogenic differ-entiation
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