Effect And Mechanism Of EID3 On Proliferation,Apoptosis And Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells

Background and purpose:The purpose of this study is to elucidate the effect of EID3 on the proliferation,apoptosis and osteogenesis of hPDLSCs,and to explore the potential regulatory mechanism of EID3,and further expand the important role of Hippo/TAZ signaling pathway in the osteogenesis of hPDLSCs.Methods:1.Isolation,culture and identification of hPDLSCs:Primary cells were obtained by tissue block adherence method,and the expression of mesenchymal stem cell markers was identified by flow cytometry.After adding osteogenic and adipogenic inducers,the ability of multi-directional differentiation was detected and the ability of clonal proliferation was detected by crystal violet staining.2.Construction of stable cell lines with knockdown and overexpression of EID3:the sequence with the best interference efficiency was screened for packaging lentiviral vector;hPDLSCs were infected by lentiviral infection system with green fluorescent protein and divided into knockdown control(shNC),knockdown group(shEID3),overexpression control(oeNC)and overexpression group(oeEID3);The efficiency of EID3 knockdown and overexpression was verified by q-PCR and Western blot analysis.3.Effects of knockdown and overexpression of EID3 on proliferation,apoptosis and osteogenic differentiation:the proliferation activity of cells was detected by EdU staining and Clonal formation assay;the effects of knockdown and overexpression of EID3 on cell cycle and apoptosis were analyzed by flow cytometry;Western blot was used to detect the expression of EID3.After osteogenic induction,the differences of early and late osteogenic ability were determined by alkaline phosphatase and alizarin red staining and alkaline phosphatase activity test.4.EID3 regulates the osteogenic differentiation of hPDLSCs through Hippo/TAZ pathway:stable cell lines with knockdown and overexpression of TAZ were constructed.The expression differences of EID3 and osteogenic related genes and proteins between knockdown and overexpression of TAZ were verified by q-PCR and Western blot.Overexpression of TAZ was induced by BMS to up regulate the expression of EID3 and verify the expression differences of osteogenic related genes and proteins.Results:1.The primary cells of hPDLSCs were successfully obtained by tissue block adherent method,which expressed the surface markers of mesenchymal stem cells and had the potential of osteogenic and adipogenic differentiation and clone formation,indicating that hPDLSCs were successfully obtained and cultured.2.The results showed that the interference effect of siEID3-416 sequence was the best,and the optimal infection condition of EID3 knockdown and overexpression lentivirus was MOI=20.The results of q-PCR and Western blot showed that we successfully constructed the stable cell lines with down-regulation and overexpression of EID3.3.After knocking down EID3,EdU labeled proliferation cells were more than those in the control group,and flow cytometry showed that the number of cells in G2/M phase was increased.Clone formation assay showed that more cell colonies were formed and the levels of proliferation related proteins were up-regulated after EID3 knockdown.At the same time,flow cytometry showed that the proportion of early apoptosis decreased,and the levels of apoptosis related genes and proteins decreased.After osteogenic induction,osteogenic genes and proteins were up-regulated,alkaline phosphatase and alizarin red staining were significantly positive,and alkaline phosphatase activity was increased4.After overexpression of EID3,EdU labeled proliferation cells were lower than those in the control group.Flow cytometry showed that the number of cells in G2/M phase decreased,and colony formation assay showed that the number of cells formed after overexpression of EID3 decreased.The levels of proliferation related proteins were down regulated.Flow cytometry showed that the proportion of early apoptosis increased,and the levels of apoptosis related genes and proteins increased.The levels of genes and proteins related to osteogenesis were decreased,alkaline phosphatase and alizarin red staining were negative,and alkaline phosphatase activity was decreased.5.BMS can increase the content of EID3 gene and protein in hPDLSCs in a time-dependent and dose-dependent manner.6.The gene and protein levels of EID3 increased when TAZ was knocked down,but decreased when TAZ was overexpressed.After induction of osteogenic differentiation,q-PCR and Western blot showed that the levels of bone related genes and proteins in shTAZ decreased,while the content of EID3 increased.The levels of bone related genes and proteins in oeTAZ group increased,while the content of EID3 decreased.Overexpression of TAZ and addition of BMS up regulated EID3.The results showed that BMS could inhibit the up regulation of osteogenesis induced by overexpression of TAZ,which indicated that EID3 could regulate the osteogenesis of hPDLSCs through Hippo/TAZ signaling pathway.Conclusion:EID3 negatively regulates the proliferation,apoptosis and osteogenic differentiation of hPDLSCs,and the regulation of osteogenic differentiation is mediated by Hippo/TAZ.