The Function And Mechanism Of WDR5 On The Mesenchymal Stem Cells

IntroductionMesenchymal stem cells are a kind of pluripotent stem cells capable of selfreplication,clone formation and multi-lineage differentiation potential.Stem cells have multiple differentiation function,so that it has application prospect in many aspects.WDR5,as the main member of WD40 protein family,plays an important role in tissue regeneration and bone tissue development,but the role of mesenchymal stem cells is not clear.It is clarified whether WDR5 is a key target for regulating mesenchymal stem cells,and provides theoretical basis for the development of small molecular agents to promote tooth regeneration and bone regeneration.Materials and MethodsWDR5 and introduced it into mesenchymal stem cells with lentiviral infection to knock-down the WDR5 and over-expressed WDR5 by infecting stem cells from apical papilla with a retroviruses construct.WDR5 expression was confirmed using Real-time RT-PCR and Western Blot analysis.The infected mesenchymal stem cells were used to do gain-of-function study.Alkaline phosphatase activity assay(ALP),Alizarin-red staining(ARS),quantitative calcium analysis and Real-Time PCR were used to study the MSCs vitro’osteo/dentinogenic differentiation potentials.Real-time RT-PCR was used to investigate the changes of genes including osteogenic marker genes Bone sialoprotein(BSP),Osteopontin(OPN),the dentinogenic markers Dentin sialophosphoprotein(DSPP)and Dentin matrix protein 1(DMP1)and the critical transcription factor Runt-related transcription factor 2(RUNX2).Vascular regeneration and neural regeneration is also important in tissue regeneration.We investigate the changes of some genes including Angiopoietin-1(ANG-1),Vascular endothelial growth factor(VEGF)and Platelet-derived growth factor-A(PDGFA)which can regulate proliferation of vascular endothelial cells.In the case of neural differentiation of SCAPs,we detect gene expression related to neurogenic differentiation which is included Tubulin,beta 3 class-III(β-III-Tubulin)and Neural cell adhesion molecule(NCAM),Tyrosine hydroxylase(TH),Neurogenic diffierentation(Neurod)via Real-Time RT-PCR.At the same time,we observed the changes of MSCs in cell morphology under the Microscope every 3 days and performed immunofluorescence analysis to detect the expression of neural markers including β-III Tubulin and Nestin.ResultsIn the process of osteo/dentinogenic differentiation,the results of ALP、ARS quantitative analysis of calcium and Real-time PCR showed that knock-down the WDR5 enhanced Osteogenesis differentiation potentials in MSCs.Compared with the control group,the osteogenic marker gene BSP,OPN and dentinogenic markers DSPP,DMP1 were significantly reduced in the SCAPs-Myc-WDR5 group cells.Also in the induction of angiogenesis,Real-Time RT-PCR analysis results showed that the expression level of ANG-1,VEGF,PDGFA were up-regulated in the SCAPs-WDR5 sh group.In the SCAPs-Myc-WDR5 group,Real-Time RT-PCR analysis implicited that the SCAPs-Myc-WDR5 group inhibited the formation of blood vessels.We proved it in the experiment of Neurogenic differentiation and immunofluorescence staining that the higher level of WDR5 decrease the neural differentiation and the low expression of WDR5 promotes neural differentiation in the MSCs.ConclusionThe above results are illustrated that WDR5 reduce Osteo/dentinogenic differentiation capacity,Vascular differentiation capacity and Neuro differentation capacity of MSCs in vitro.It is indicate that WDR5 can provide theoretical basis and key target for total tooth regeneration.