Thy1 Promotes The Proliferation,Migration And Angiogenesis Of TECs Through The αvβ3/FAK Pathway

Background The traditional theory of tumor angiogenesis believes that the new blood vessels in tumors are formed by budding from the host blood vessels,so they are relatively stable in genotype and phenotype.However,more and more studies have confirmed that due to the long-term exposure of new blood vessels in tumors to the tumor microenvironment,changes in cell morphology,function,phenotype,and even genotype have occurred,known as tumor endothelial cells(TECs).Compared with normal vascular endothelial cells(NECs),TECs exhibit significant differences in molecular structure and function.Discovering TECs specific marker molecules and clarifying the potential role and regulatory mechanism of TECs in tumor angiogenesis will hopefully open a new window for anti tumor angiogenesis therapy.Thymus cell antigen 1(Thy1),also known as CD90(Cluster of Differentiation),is a member of the glycosylated phosphatidylinositol anchor protein(GPI-AP)family.It belongs to the immunoglobulin superfamily.Human Thy1 is expressed in nerve cells,CD34+cell subsets,fibroblasts,activated endothelial cells,and related to cell adhesion,differentiation,and intercellular interactions.Thy1 is also a marker of activation of human microvascular endothelial cells and is associated with the formation of new blood vessels.In addition,more and more studies have shown that Thy1 plays a role in regulating the proliferation,migration,and angiogenesis of cancer cells.There is also evidence that Thy1 is an important prognostic marker for many cancers.However,the mechanism by which Thy1 functions is unclear.Integrins are a class of non covalently related heterodimeric transmembrane glycoprotein adhesion molecules that are expressed on the surface of endothelial cells and pericytes.The composition of heterodimers endows them with ligand specificity.Most integrins can mediate cell to ECM and cell to cell interactions by recognizing extracellular matrix(ECM)proteins.among α v β Three of them were expressed in endothelial cells and could not be detected in resting blood vessels,but their expression was significantly upregulated in neovascularization.In this study,tumor endothelial cells were used as the entry point,and transcriptome sequencing was performed to obtain the highly expressed Thy1 molecule specific to TECs.Through GO and KEGG functional annotation and enrichment analysis,it was found that Thy1 is a key molecule in the signal regulatory network of immune cells and tumor angiogenesis in the tumor microenvironment.Thy1 activated on the surface of TECs may be related to α v β Integrin specific binding promotes tumor angiogenesis.The clarification of this mechanism will provide a new way to design new anti tumor angiogenesis therapies targeting Thy1.Research purposeExplore Thy1 Pass α v β3 The role and possible mechanisms of FAK in promoting the proliferation,migration,and angiogenesis of TECs provide theoretical guidance and experimental evidence for anti vascular therapy targeting TECs.Methods1.Nanopore full-length transcriptome sequencing and differential gene analysis were performed on TECs 2H-11 and NECs SVEC4-10 cells,and the differential genes were screened according to their angiogenic function to obtain a differential gene Thy1 that was significantly overexpressed in 2H-11.Further,the GO function and KEGG pathway enrichment analysis of differential genes related to angiogenesis was conducted,focusing on the function and pathway regions enriched by Thy1.To verify the sequencing results,Western blot,RT-PCR,and cellular immunofluorescence assay were used to detect the expression of Thy1 protein and m-RNA in 2H-11 and SVEC4-10,respectively,and the localization of Thy1 in cells.2.To explore the relationship between Thy1 and α v β Whether the two bind in 2H-11 is detected by cell immunofluorescence double staining,respectively α v β 3.Whether there is co localization and Co-IP experiment to detect Thy1 and α v β 3.Protein binding and String database observation α v β The relationship between the two.3.Using sh RNA lentivirus transfection to downregulate the expression of Thy1 in 2H-11 cells,RT-PCR and Western blot were used to detect the transfection efficiency of Thy1.Then,the down-regulated Thy1 group and the control group were used to conduct CCK8 experiment to detect cell proliferation ability,scratch experiment,transwell chamber experiment to detect cell migration ability,and in vitro tubular formation experiment to detect cell tubular formation ability.Western blot detected that after downregulation of Thy1,2H 11 β Changes in the expression of FAK and p-FAK proteins are intended to clarify that Thy1 may pass through α v β The 3/FAK pathway promotes the proliferation,migration,and in vitro tubular ability of TECs.Results1.Transcriptomic analysis and screening of Thy1 for 2H-11 and SVEC4-10According to functional analysis,47 differentially expressed genes related to angiogenesis were selected from the transcriptome sequencing results of 2H-11 and SVEC4-10.After ranking by multiple of differences,Thy1 ranked second.Thermograms can visually show that Thy1 was significantly overexpressed in three tumor endothelial samples and significantly underexpressed in three normal endocutaneous samples,with a significant difference between the two.2.Enrichment analysis of angiogenic related differential gene Thy1GO function analysis shows that Thy1 has different enrichment trends in all genes and differential genes under immune regulation,cell movement,and adhesion.It is important to analyze whether these three functions are related to differences in angiogenesis.KEGG pathway analysis shows that Thy1 is enriched in the leukocyte transendothelial migration pathway.In the pathway diagram,it can be seen that the downstream of Thy1 mainly includes signal molecules such as Rho A and FAK.RTPCR and Western blot analysis showed that the expression of Thy1 m RNA and protein in 2H-11 was significantly higher than that in SVEC4-10(P<0.0001).The above results demonstrate that compared to SVEC4-10,Thy1 is significantly overexpressed in 2H-11,consistent with the sequencing results obtained by transcriptomics.3.Thy1 in 2H 11 can be compared to α v β 3 Combine with each otherIF experiments confirm that Thy1 can match α v β There is a co localization phenomenon in the cytoplasm and cell membrane.String database displays Thy1 and β 3(ITGB3)has protein interaction.Co-IP experiments further confirmed that Thy1 in 2H-11 can be compared to α v β 3 can be combined with each other.The above results indicate that Thy1 in tumor endothelial cell 2H-11 can interact with α v β 3 Combine with each other.4.Effect of Thy1 on the proliferation,migration,and tubular formation ability of tumor endothelial cells in vitro(1)Construction and identification of a 2H-11 cell line downregulated by lentivirus transfection: Western blot and RT-PCR results showed that the protein and m RNA expression of Thy1 in the downregulated group was significantly lower than that in the control group(P<0.05,P<0.01).The results showed that a TECs 2H-11 cell line that downregulated the expression of Thy1 was successfully constructed.(2)The effect of downregulation of Thy1 on the proliferation,migration,and in vitro tubular formation of 2H-11: After downregulation of Thy1,the proliferation ability of cells decreased,manifested as a significant decrease in cell viability on the 3rd and 4th days(P<0.05)and(P<0.01);The decrease in cell migration ability was manifested by a decrease in cell migration rate(P;The ability of cells to form tubules in vitro was reduced,as evidenced by a decrease in the total length of the ring-shaped branches of cells after downregulation of Thy1,which was longer than that of the control group after 4 hours(P<0.001).The above results demonstrate that after downregulation of Thy1 expression,the proliferation,migration,and in vitro tubular formation abilities of 2H-11 are reduced.(3)Thy1 passed α v β The 3/FAK pathway promotes the proliferation,migration,and in vitro tubular formation of TECs: Western blot detected that after downregulation of Thy1 β The results showed that downregulation of Thy1 inhibited the expression of FAK and p-FAK proteins β Expression of FAK and p-FAK proteins.(*,P<0.05;**,P<0.01)。 The above results prove that Thy1 may pass α v β The 3/FAK pathway promotes the proliferation,migration,and angiogenesis of TECs.