TAZ Promotes The Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells Through The TGF?/SMAD3 Signalling Pathway

Objective:The purpose of this study was to investigate the role and mechanism of HIPPO/TAZ signaling pathway in the proliferation,dry maintenance and osteogenic differentiation of human periodontal ligament stem cells.Methods:1.Human periodontal ligament stem cells were obtained by enzyme digestion.The stem markers(CD90,CD44,CD 105,CD73,etc.)were identified by flow cytometry to detect whether they had osteogenesis,adipogenic and chondrogenic differentiation ability.2.Human periodontal ligament stem cells were infected with green fluorescent protein lentivirus infection system,grouped into knockout control(shNC)and knockout group(shTAZ),overexpression control(Vector)and overexpressed TAZ group(TAZ+).The efficiency of TAZ knockdown and TAZ overexpression was examined by Western blot and qRT-PCR,respectively.3.The cell proliferation activity of each group was detected by EdU staining.The proliferation of the control group and the experimental group was detected by CCK-8 for 7 days.Whether the change of TAZ caused the proliferation-related signaling pathway(PI3K/AKT,ERK,etc.)was detected by Western blot.4.Flow cytometry analysis of the difference in apoptosis of human periodontal ligament stem cells overexpressing TAZ and knocking out TAZ;the changes of apoptosis-related proteins(CASPASE3,BCL2,BAX)were detected by Western blot.5.Western blot and RT-PCR were used to detect the difference of expression levels of cell dry maintenance related proteins and genes(NANOG,SOX2,OCT4,etc.).6.Simultaneous osteogenic induction of each group of cells,quantitative detection of alkaline phosphatase activity caused by TAZ knockout and overexpression by alkaline phosphatase staining and alkaline phosphatase activity;induction of 21 days,alizarin red staining The differences in the growth of mineralized nodules between the groups were determined.Western blot and RT-PCR were used to detect the expression of osteogenic related genes and proteins(COL1,ALP,RUNX2,etc.)in each group at 7 days.7.SIS3 HCL,a chemical inhibitor of SMAD3,was used to overexpress the TAZ group in h-PDLSCs.Compared with the overexpressed TAZ group,alizarin red staining was used to detect osteogenic differentiation and bone formation in vitro;Western-blot was used to detect osteogenesis.Expression of related proteins(ALP,RUNX2,COL 1,etc.).Results:1.Successfully isolated and purified human periodontal ligament stem cells,and successfully constructed over-expressed TAZ and knockout TAZ cell models.2.Overexpression of TAZ increased the proliferative capacity of human periodontal ligament stem cells and attenuated late apoptosis;and overexpression of TAZ caused up-regulation of p-AKT and p-ERK expression.3.After knocking down TAZ,the proliferative activity of h-PDLSCs decreased and the rate of late cell apoptosis increased;the expression levels of p-AKT and p-ERK were down-regulated.4.Overexpression of TAZ group dry maintenance related genes and protein expression levels up-regulated,knockout TAZ group dry maintenance related genes and protein expression levels down-regulated.5.After 7 days of osteogenic induction,the alkaline phosphatase activity of the overexpressed TAZ group was significantly increased,and the alkaline phosphatase activity of the TAZ group was significantly decreased.The osteogenic induction was significant for 21 days,and the overproduction of the TAZ group was significant.increase.Furthermore,after 7 days of osteogenic induction,the expression of osteogenic proteins and mRNA levels in the overexpressed TAZ group was up-regulated,and the osteogenic associated protein and mRNA expression levels in the TAZ group were down-regulated.6.Osteogenesis induction for 21 days,compared with the over-expressed TAZ group,after SIS3 HCL(1uM),alizarin red staining,mineralization nodule formation was significantly down-regulated,and osteogenic related proteins(ALP,RUNX2,COL1)and other expression level down.Conclusion:HIPPO/TAZ plays a positive role in the proliferation,dry maintenance and osteogenic differentiation of h-PDLSCs,and this regulation is mediated by TGF?/SMAD3.