PoFUT1 Promotes Placental Angiogenesis By Increasing O-fucosylation Of UPA In Trophoblast Cells

Ⅰ.Background and Objective:Embryo implantation is a complicated biological process that requires the simultaneous establishment of mature embryos and accepted endometrium.During embryo implantation,the trophoblast on the surface of the blastocyst mediated the recognition between the embryo and the endometrium,promoting embryo implantation,placental development,and maintaining a successful pregnancy.During normal pregnancy,trophoblast cells are able to degrade the extracellular matrix,invade the maternal endometrium and blood vessels,and establish a connection between the mother and fetus.Deficiency of trophoblast proliferation and invasion can impede embryo implantation,leading to miscarriage,and pregnancy-related diseases such as fetal intrauterine growth restriction(FIGR)and preeclampsia.Protein O-fucosyltransferase 1(poFUT1)is a key enzyme that catalyzes the synthesis of protein O-fucose.The previous study of the research group showed that compared with normal early pregnancy,the expression of poFUT1 in trophoblast cells was significantly decreased in abortion patients;poFUT1 promoted the invasion and proliferation of trophoblast cells.However,the role and mechanism of poFUT1 in the involvement of trophoblast cells in placental vascular remodeling has not been reported.This article aims to investigate the role of poFUT1 in trophoblast cells in placental angiogenesis and its mechanism,which will help to understand the mechanism of embryo implantation and diagnose the pregnancy-related diseases such as glyco-targeted abortion.And treatment provides a new theoretical basis.Ⅱ.Method: 1.Expression of Cytokeratin 7,poFUT1 and angiogenesis marker CD34 in villi of normal early pregnancy and patients with miscarriage by immunohistochemical staining.Expression of HLA-G and CD34 in decidua tissues of normal early pregnancy and patients with miscarriage by immunohistochemical staining.2.Real-time PCR,western blot were used to detect the transfection efficiency of poFUT1 cDNA and poFUT1 siRNA;transwell and F-actin were used to detect the migration and the ability to rearrange the cytoskeleton of trophoblast cells;gelatin zymography was used to detect the secretion of MMPs in the supernatant;tube-forming experiments were performed to analyze the ability of cells to form tubes;western blot was used to detect the expression of angiogenesis-related proteins.3.O-Fucose kit detects total O-fucose expression on the surface of trophoblast cells;co-immunoprecipitation(co-IP)detects the expression of O-fucose on urokinase-type plasminogen activator(uPA);the expression of uPA in culture medium was detected by plasminogen zymography.4.Transfected poFUT1 cDNA,poFUT1 mutant,uPA cDNA,uPA mutant into trophoblast cells(HTR-8/SVneo)by Lipofectamine 2000,western blot was used to detect cell cycle proteins;transwell was used to detect cell migration ability;the ability of the cell tube formation was analyzed by Matrigel tube-forming experiment;uPA binding to uPAR was detected by uPAR immunoprecipitation;western blot analysis of the activation of RhoA signaling pathway in trophoblast cells.5.The supernatant of the trophoblastic treatment group was collected to treat human umbilical vein endothelial cells(HUVEC),and the expression level of cell cycle proteins were detected by western blot;cell migration and cytoskeleton rearrangement were detected by transwell and F-actin;the ability of the two cells to form a tube together was verified by Matrigel tube-forming experiments;western blot was used to detect the activation of RhoA signaling pathway in HUVEC.6.Chicken chorioallantoic membrane(CAM)was verified in vivo: human chorionic villus tissue was collected and treated with poFUT1 siRNA(50 nM)and then applied to CAM to observe the changes of blood vessels on CAM.Ⅲ.Results: 1.The samples of villus and decidual tissue from normal early pregnancy and miscarriage were collected for immunochemical staining.The results showed that compared with normal early pregnancy,the expression of poFUT1 in miscarriage tissue was significantly reduced,and the spiral artery lumen was small.The expression of HLA-G in decidua was decreased and the diameter of spiral artery was significantly smaller than that of normal early pregnancy.2.poFUT1 promotes angiogenesis in trophoblast cells.The results of transwell and F-actin showed that compared with the control group,the migration ability of trophoblast cells was significantly enhanced by transfection of poFUT1 cDNA,while the transfection of poFUT1 siRNA inhibited the migration of trophoblast cells.The expression of matrix metalloproteinases(MMP2,MMP9)in culture medium was determined by gelatin zymography.The results showed that the secretion of MMP2 and MMP9 increased after transfection of poFUT1 cDNA.The results of tube-forming experiments showed that the expression of trophoblastic cells was significantly enhanced in the transfected poFUT1 cDNA group,and the expression of vascular-related markers CD31,CD34 and angiogenesis-related proteins(VE-cadherin,VEGFR2,uPA)was significantly increased.3.poFUT1 increases the biosynthesis of O-fucose on uPA.O-Fucose results showed that after transfection of poFUT1 siRNA,the total O-fucose expression of trophoblast cells was decreased,and the poFUT1 siRNA+poFUT1 cDNA group could up-regulate the expression of O-fucose.The results of co-immunoprecipitation of the protein showed that the expression of O-fucose on uPA was increased in the transfected uPA cDNA group,while the O-fucosylation level of the trophoblast was higher in the transfected uPA mutant group than in the untransfected group.No significant changes were observed.The results of plasminogen zymography showed that the secretion of uPA was increased in the culture medium by transfecting the poFUT1 cDNA group.4.poFUT1 promotes proliferation,migration and angiogenesis of trophoblast cells by increasing O-fucosylation of uPA.Western blot analysis of cell cycle proteins showed that transfection of poFUT1 cDNA and uPA cDNA promoted the proliferation of trophoblast cells.Transwell and tube-forming experiments showed that the migration and tube-forming ability of trophoblast cells were significantly enhanced by transfection of poFUT1 cDNA and uPA cDNA.The results of IP experiment with uPA-specific receptor uPAR showed that the number of uPA binding to uPAR increased in the transfected poFUT1 cDNA,uPA cDNA group,and the uPA transfected with poFUT1 mutant and uPA mutant,and uPAR had no significant change compared with the untransfected group.Western blot results of RhoA signaling pathway showed that the expression of RhoA-GTP,p-LIMK1 and p-cofilin was enhanced in the transfected poFUT1 cDNA and uPA cDNA groups.Addition of RhoA signaling pathway inhibitor(C3 transferase)can inhibit the phosphorylation level of the above proteins.5.The culture medium of the trophoblast cells treated with different treatments was applied to human umbilical vein endothelial cells(HUVEC).Western blot results showed that the secretion of trophoblast cells in the poFUT1 cDNA transfection group can promote the proliferation of endothelial cells.The results of transwell and F-actin showed that the secretion of trophoblast cells in the poFUT1 cDNA transfection group can promote the migration of endothelial cells and promote the rearrangement of endothelial cytoskeleton.The results of tube-forming experiments showed that the tube-forming ability of endothelial cells was significantly enhanced after treatment with the secretion of trophoblast cells in the poFUT1 cDNA transfection group.Western blot analysis of RhoA signaling pathway showed that the secretion of trophoblast cells in the poFUT1 cDNA transfection group promoted the expression of RhoA-GTP,p-LIMK1 and p-cofilin in endothelial cells.6.The in vivo experiment of chicken chorioallantoic membrane showed that the capillary distribution and congestion of the allantoic membrane were significantly enhanced in the plush tissue treatment group compared with the PBS-treated group.When the villus tissue was treated with poFUT1 siRNA,the vascular branch was reduced and the hyperemia was reduced compared with the untreated villus group.Ⅳ.Conclusion: 1.poFUT1 promotes the proliferation and migration of trophoblast cells and promotes angiogenesis in trophoblast cells.2.poFUT1 increases the interaction with its receptor uPAR by up-regulating the synthesis of O-fucose on uPA,and promotes the formation of angiogenesis.3.poFUT1 activates the RhoA signaling pathway by up-regulating O-fucosylation on uPA,further enhancing angiogenic capacity.