Transcription Factor SOX2 Promotes Human Umbilical Vein Endothelial Cell Proliferation Through JAGGED1

BackgroundAngiogenesis,the formation of new blood vessels from pre-existing vessels,is a critical process in development,tissue regeneration and it is also associated with a variety of diseases,such as tumor.and rheumatoid arthritis.Angiogenesis includes multiple progresses and kinds of cells involve in it,while the changes of endothelial cells are the most important.The endothelial cells activated by factors released from hypoxia cells,such as vascular endothelial growth factor,that leads to angiogenesis.The process requires a unique specialized tip cell to explore the surrounding environment and guide the direction of sprouting,and adjacent cells specialize into stalk cells through mutual signals between cells and follow tip cell to proliferate,finally become tube lumen.Then the adjacent tubes fuse and connect with each other,covering the peripheral cells and perfuse into mature blood vessels.The negative feedback regulatory loop formed by the VEGF and NOTCH pathway plays an important role in the selection and maintenance of tip/stalk cells.Our previous analysis of transcription network in tip/stalk cell phenotype indicates that SOX2 is in the position of key nodes in the transcription network of the two cell phenotypes,which suggests that SOX2 may play a key role in angiogenesis.We select human umbilical vein endothelial cells(HUVEC)in the previous experiment as the research object,for it can represents external physiological and toxicological response characteristics of the majority of vascular endothelial cells in human body,and it has been a classic model for many researchers.Our previous experiments demonstrate that overexpression SOX2 in HUVEC can promote its proliferation.We hope to further clarify the molecular mechanism of transcription factor SOX2 in promoting proliferation,which can enrich the mechanism of sprouting angiogenesis.Method:The pcDNA3.0-SOX2 plasmid was transfected into HUVEC by liposome,and G418 was used to select.Then,the SOX2~+HUVEC was constructed.qPCR was used to detect the transcription level of SOX2,and the protein level of SOX2 was detected by Western blot.Then,qPCR was used to identify the transcription level of NOTCH and VEGF pathway related ligands and receptors,and the differentially expressed genes were selected out preliminarily.Western blot was used for further validation and selection.Chromatin immunoprecipitation(Ch IP)is a method of studying the interaction between DNA and proteins,and it was used to determine whether the target gene was direct regulated by SOX2.Then the direct target gene was silenced in the SOX2~+HUVEC line,and the proliferative capacity was measured by CCK8,Ed U and flow cytometry.Result:The pcDNA3.0-SOX2 plasmid was transfected into HUVEC by liposome,and G418 was used to obtain 7 monoclonal cell lines(named SOX2-n,n=1~7).In contrast with normal HUVEC and transfection control group(empty vector,EV),4 of them had higher expression,there is significant difference(P<0.05).Western blot was used to identify the top two lines,which named SOX2-1,SOX2-2 for subsequent experiments,and these two lines had higher level of expression of SOX2(P<0.001,P<0.01).qPCR was used to detect expression of receptors and ligands in VEGF pathway.The results showed that in SOX2-1,the expression of NRP1 significant decreased(P<0.05),there was no significant difference in VEGFR1-3 and VEGFA(P>0.05).There was no significant difference genes in SOX2-2(P>0.05).Then qPCR was used to detect the differential expression of ligands and receptors in NOTCH pathway.Significant decreased expression of DLL4 and increased expression of JAGGED1 was detected in SOX2-1(P<0.05),while DLL1,DLL3,JAGGED2,NOTCH1,NOTCH2,NOTCH3,NOTCH4 have no significant difference(P>0.05).In SOX2-2 cell lines,the expression of JAGGED1 was increased with statistical significance(P<0.05),while JAGGED2,NOTCH1,NOTCH2,NOTCH3,NOTCH4 had no statistical difference(P>0.05).Western blot was used to detect the protein levels of DLL4,NRP1,JAGGED1 in SOX2-1 and SOX2-2.The results showed that the expression of JAGGED1 in SOX2-1 and SOX2-2 was higher than that in EV,and the difference was statistically significant(P<0.05,P<0.01).The expression of DLL4 was lower than that in EV(P<0.05).The expression of NRP1 was increased in SOX2-2(P<0.05)while it had no difference in SOX2-1(P>0.05).We found no correlation between DLL4 and SOX2 in literatures.So Ensembl database was used to search for the sequence of the promoter region of NRP1,DLL4,JAGGED1,and the Jaspar database was used to predict the SOX2 binding site on line.The results showed that JAGGED1 had a binding site,NRP1 had two binding sites,while DLL4 did not find a possible binding site.Therefore,DLL4 does not verificated by Ch IP-qPCR.Using Ch IP-qPCR to verify if SOX2 direct binding to the promoter of selected genes NRP1,JAGGED1,and the results showed that SOX2 antibody group significantly enriched more JAGGED1 promoter fragments than Ig G group,the result was statistically significant(P<0.01),while NRP1 had no statistically significant differences between the SOX2 antibody group and Ig G group(P>0.05).These results suggest that SOX2 can directly bingding the promoter region of JAGGED1 to regulate its expression.We had proved SOX2 can promote HUVEC proliferating in our previous study.JAGGED1 is closely related to cell proliferation,so we focus on its ability promoting proliferation of cells.Knock down JAGGED1 by three lentivirus sh RNA-JAGGED1 sequence in SOX2-1 respectively.The cell with e GFP was 80%-90% in control group(SOX2~+/Scrambled sh RNA)and the silence group.Subsequently,qPCR and Western blot was used and the expression of JAGGED1 was significantly decreased in the silence group especially the third silence group,so it was selected for the follow-up experiments.Then the proliferation of cells was detected by CCK8 assay and Ed U assay.The results show that cell proliferation decreased in SOX2~+/JAGGED1-group than in the SOX2~+/Scr sh RNA group,with statistical significance.And its population doubling time was longer than the control group(P<0.05).Cell cycle and cell proliferation were calculated by flow cytometry,and the same result was obtained(P<0.05).These results indicate that the silencing JAGGED1 can counteract the proliferation by SOX2 overexpression.This suggests that in HUVEC,the transcription factor SOX2,at least in part,promotes cell proliferation by up regulated its direct target gene JAGGED1.Conclusion:In HUVEC,SOX2 can up-regulate the expression level of JAGGED1,and it can directly binding to the JAGGED1 promoter region.Silencing JAGGED1 can partially block the effect of SOX2 on cell proliferation.