The Effect And Mechanism Of H3K27me3 In Endothelial Cell Reprogramming By SOX2

Background:Endothelial progenitor cells are significant to the treatment of angiogenesis,endothelial dysfunction and other related cardiovascular pathological changes and vascular diseases.However,endothelial progenitor cells have limited sources and reprogramming endothelial cells is an effective method for obtaining endothelial progenitor cells.The four reprogramming factors OCT4,SOX2,KLF4 and c-MYC are able to "reprogram" cells,allowing cells to break through the epigenetic barrier and gain pluripotency.H3K4me3 and H3K27me3 can promote and inhibit the transcription of genes and the bivalent modification of the two maintains the expression of the gene at a lower level of equilibrium.Objective: Explore the role of SOX2 in endothelial cell reprogramming and the mechanism of histone methylation in this process.Methods: In this study,human umbilical vein endothelial cells were used as the research object.In the overexpressed SOX2 group,the empty vector group and the human umbilical vein endothelial cells,RT-q PCR and western blot detected the expression of endothelial progenitor cell markers CD133,CD34,VEGFR2 and v WF at the gene expression and protein levels;we observed ultrastructural changes in cells by transmission electron microscopy;we also observed the degree of low-density acetyl-lipoprotein intake;the effect of tube-forming ability was determined by Matrigel tube assay;Ed U and the colony formation assay displayed the proliferative capacity of the cells;Comparison of pc DNA3.0-SOX2 and pc DNA3.0,differentially expressed genes were detected by RNA-seq,and they were analyzed by cluster analysis,functional annotation and enrichment analysis;the change of H3K27me3 and H3K4me3 was detected by western blot and immunofluorescence;the change of H3K27me3 modified enzyme EZH2 was detected by RT-q PCR and western blot;finally,the bioinformatics was used to predict the binding site of SOX2 in the EZH2 promoter region.Results: After overexpressing SOX2,RT-q PCR indicated that the expression of CD133,CD34,v WF and VEGFR2 in endothelial progenitor cells was up-regulated.western blot indicated that the expression of CD133,CD34,v WF was up-regulated,however,the expression of VEGFR2 was decreased.Transmission electron microscopy showed that the cells had more chromatin and decreased heterochromatin,the nucleolus became loose,some nucleoli were spongy,the number of nucleoli increased,the mitochondria became thicker,the mitochondrial membrane became thicker and the number of phagocytic vacuoles increased;Low-density acetyl-lipoprotein uptake showed low-density acetyl-lipoprotein uptake decreasing;Matrigel tube assay showed angiogenic capacity reducing;Ed U and clone formation experiments demonstrated no effect on proliferative capacity;Cluster analysis showed that the expression of 106 differentially expressed genes and the functional correlation of similar expression patterns,subsequent functional enrichment analysis showed that differentially expressed genes are involved in the regulation of multicellular organismal development,regulation of cell development,regulation of developmental process,regulation of cell differentiation and other biological processes and some signaling pathways such as regulating pluripotency of stem cells,functional annotation analysis show that differentially expressed genes are associated with developmental process,organelle,membrane,chromatin structure,dynamics,translation,ribosomal structure and biogenesis,transcription.western blot and immunofluorescence suggested that the expression of H3K4me3 was unchanged and the expression of H3K27me3 was down-regulated after overexpression of SOX2,suggesting that the change of H3K27me3 expression may affect the regulation of SOX2 on endothelial cell dedifferentiation;RT-q PCR and western blot showed that H3K27me3 methylase EZH2 decreaseing at gene expression and protein levels;Finally,SOX2 may have five binding sites in the promoter region of EZH2 by bioinformatic analysis.Conclusion: In human umbilical vein endothelial cells,SOX2 down-regulates H3K27me3 by down-regulating histone methylase EZH2,and may play a transcriptional regulatory role by directly binding to the EZH2 promoter region.Overexpression of SOX2 activates transcription of genes such as endothelial progenitor cell marker CD133;low-density acetyl-lipoprotein uptake and tube-forming ability are reduced,which suggest that dedifferentiation may occur,obtaining endothelial progenitor cells or cells with characteristic of endothelial progenitor.