Regulation Of Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells By DNA Methylation Modification Of Dishevelled Protein

Bone marrow mesenchymal stem cells(BMSCs)are considered to be one of the most promising therapeutic cell sources as they encompass a plasticity of multiple cell lineages.The challenge in using these cells lies in developing well-defined protocols for directing cellular differentiation to generate a desired lineage,thus orienting its differentiation direction.In addition to the traditional transcription factor regulation,epigenetic modification also plays an important role in the regulation of bone marrow mesenchymal stem cells differentiation.DNA methylation is one of the main ways of epigenetic regulation.Dishevelled(Dsh)is a family of proteins that deals with typical and atypical Wnt signaling pathways.Dsh is a cytoplasmic phosphoprotein that acts directly on the downstream of the crimp receptor,which plays an important role in both embryos and adults,and affects the whole process from cell differentiation to social behavior.Research purposesIn this paper,by using osteoblast differentiation of BMSCs a research model,the effect of DNA methylation of Dishevelled protein on osteogenic differentiation of BMSCs was studied by DNA methylation inhibitor(5-azacytidine),and explored the potential effect of methylation modification on differential gene expression.Research methods(1)Human bone marrow mesenchymal stem cells induced differentiation in osteogenic medium,and the degree of differentiation of stem cells was identified by alizarin red staining.AKP kit was used to detect the activity and expression of alkaline phosphatase during osteogenic differentiation.The expression level of Dishevelled protein was seen by the way of RT-PCR and Western blot.(2)The methylation of Cp G island in the promoter region of Dishevelled protein gene was detected by methylation specific PCR(MSP).(3)The expression levels of Wnt,GSK3,Axin,Dishevelled and ?-catenin were detected by RT-PCR and Western blot before and after treatment with methyltransferase inhibitor 5-aza-2'-deoxycytidine.(4)The Cp G island methylation of Dishevelled promoter was using specific methylatedtransferase,and carried out by high-throughput sequencing analysis of transcriptional cells to analyze the expression of differentially expressed genes.(5)Human bone marrow mesenchymal stem cells were treated with 5-Aza-2'-deoxycytidine,a methyltransferase inhibitor,and the expression of PP2 A was detected by RT-PCR and Western blot.(6)The mimics and inhibitors of PP2 A were synthesized and transfected into humanbone marrow mesenchymal stem cells.The expression of PP2 A gene was detected by RT-PCR and Western blot.The differentiation of stem cells was identified by alizarin red staining.Research results(1)With the bone marrow mesenchymal stem cells in osteogenic medium induced differentiation time,the greater the degree of osteogenic differentiation,while the alkaline phosphatase and Dishevelled protein expression also showed a rising trend.(2)The degree of methylation of Cp G island in the promoter region of bone marrow mesenchymal stem cells decreased with the induction of differentiation after 7,14,and 21 days after induction.(3)With the addition of methylase inhibitor-5-aza-2'-deoxycytosine to act on osteogenic differentiation of mesenchymal stem cells,compared with the DMSO control group,with the induction of differentiation time increased,Wnt,GSK3,Axin,Dishevelled and ?-catenin showed an increasing trend.(4)After high-throughput analysis of human bone marrow mesenchymal stem cells transfected with methylated promoter,PP2 A was found to be abnormally expressed in differentiated cells.It was further confirmed by regulation of expression experiments that overexpression of PP2 A gene can promote proliferation of mesenchymal stem cells Differentiation.ConclusionThis article irrefutablely demonstrated that the regulation of Cp G island methylation in the promoter region of Dishevelled protein can regulate the differentiation of mesenchymal stem cells by regulating the expression level of regulatory factor PP2 A in the regulation of BMSCs differentiation.Stem cells in osteogenic differentiation and application of the study provided an important basis and experimental basis.