Fucosyltransferase Ⅳ(FUT4) Promotes The Process Of Endometrial Decidua Function And Mechanism Research

Objective In mammalian reproduction,endometrial decidualization is the process of embryo implantation into the endometrium,which causes extensive remodeling of the endometrial stroma.Endometrial decidualization is a very important process for embryo implantation.Abnormal endometrial decidualization can cause many pregnancy-related diseases,such as infertility,recurrent miscarriage,intrauterine growth restriction,preeclampsia,and endometriosis.Protein glycosylation is a common way of protein post-translational modification,divided into N-glycosylation and O-glycosylation.Fucosylation is an important glycosylation modification method.Fucosylation is involved in important physiological and pathological processes such as embryonic development,tumorigenesis and immune dysfunction.Fucosyltransferase is a key enzyme that catalyzes fucosylation.It is divided into N-salinofucosyltransferase and O-fucosyltransferase.It catalyzes the transferation of fucose groups from nucleoside diphosphate fucose to accecp molecule.N-fucosyltransferase IV(fucosyltransferase 4,FUT4)catalyzes the linking of fucose(L-fucose)to H type 2 [Fuc → 1Galβ1 →4Glc NAcβ1→R] antigen via α-1,3 glycosidic bond to produce LeY(Lewis antigen Y,LeY)[Fuc1 → 2Galβ1 → 4(Fuc1 → 3)Glc NAcβ1 → R].LeY is a Lewis oligosaccharide on the cell surface.LeY is expressed stage-specifically during embryonic development.It is highly expressed on the surface of the embryo during the implantation window and participates in the embryo implantation process.Preliminary laboratory studies have shown that LeY on the surface of endometrial epithelial cells mediates the adhesion of embryos and endometrial epithelium,but the effect and mechanism research on the decidualization of uterine stromal cells has not been reported yet.Method1.Use immunohistochemical experiments to detect the expression of FUT4/LeY/LTL in the endometrial tissue during the proliferative,secretory,normal early pregnancy and inevitable abortion periods.2.Induce human endometrial stromal cells in vitro,and use Western blot,q RT-PCR and cellular immunofluorescence experiments to detect the expression of post-decidual decidual markers and FUT4 in stromal cells.3.The expression of FUT4 in h ESCs cells was regulated by transfection of FUT4-si RNA and FUT4-c DNA,and the expression of FUT4 and decidual markers was detected by Western blot and q RT-PCR.4.Adjust the expression of FUT4 in h ESCs cells by transfection with FUT4-si RNA and FUT4-c DNA,and use Western blot,CCK8 and cell immunofluorescence experiments to detect cell proliferation after decidualization.5.Screen and determine LeY’s target protein CD47 through pull down experiment and mass spectrometry.After detecting decidua through immunoprecipitation experiment,regulate FUT4 to detect changes in LeY on specific target proteins and changes in the signal pathway mediated by them.The effect on the decidual process.Result1.Immunohistochemical detection of the expression of FUT4/LeY in human tissues during proliferation,secretion,early pregnancy and inevitable abortion.The results show that the expression of FUT4/LeY in the proliferation,secretion and early pregnancy increases sequentially,but in the inevitable abortion The expression of period is lower than the first trimester.2.After decidualization of stromal cells is induced in vitro,the cell morphology becomes larger,from a spindle shape to a round shape,the expression of decidual markers PRL and IGFBP-1 increases,and the expression of FUT4 in h ESCs cells increases after decidualization.3.FUT4 promotes decidualization of stromal cells.Through Western blot,q RT-PCR and cellular immunofluorescence experiments,the results showed that h ESCs induced decidualization while down-regulating the expression of FUT4,which could inhibit the expression of decidual markers PRL and IGFBP-1.Up-regulate FUT4 and promote the expression of PRL,IGFBP-1 and FUT4.4.FUT4 promotes the proliferation of endometrial stromal cells by activating cyclin.Cellular immunofluorescence results showed that,compared with the control group,the expression of proliferating nuclear antigen(PCNA)in h ESCs cells transfected with FUT4-si RNA decreased;after transfection with FUT4-c DNA,the expression of PCNA was higher than that in the control group.The results of Western blot experiments showed that,compared with the control group,FUT4-si RNA transfection reduced the expression of cell proliferating nuclear antigen(PCNA)and cyclin(CDK4,CDK6);transfected FUT4-c DNA,cell proliferating nuclear antigen(PCNA))And the expression of CDK4 and CDK6 increased.The results of CCK-8 experiments showed that FUT4-si RNA inhibited cell proliferation;FUT4-c DNA promoted cell proliferation.5.FUT4 activates the PDK/AKT/GSK-3β signaling pathway by up-regulating the expression of LeY to promote endometrial decidualization.Pass the results of the pull down experiment and perform mass spectrometry analysis.Mass spectrometry results screened that there are 233 proteins that are significantly differentially expressed in normal early pregnancy compared with inevitable abortion.The target protein CD47 was further screened from GO function.By immunoprecipitation experiment,compared with the control group,when transfected with FUT4-si RNA,the expression of LeY on CD47 was reduced,the signal pathways of p-PDK,p-AKT,and p-GSK-3β were inhibited,and the decidua was inhibited;Transfection of FUT4-c DNA,compared with the control group,the expression of LeY on CD47 increased,and the p-AKT,p-PDK and p-GSK-3β signaling pathways were activated to promote decidua.After adding pathway inhibitor LY294002,the expression of decidual markers PRL and IGFBP-1 decreased.In conclusion1.Compared with the first trimester,the expression of FUT4/LeY is reduced in the tissues of patients with inevitable abortion.2.FUT4 increases the synthesis of LeY on CD47,activates the PDK/AKT/GSK-3βsignaling pathway,and promotes decidualization of the uterine stroma.