Epiregulin Up-regulates Protein O-fucosyltransfe Rase 1(poFUT1)and O-fucosylation And Promots Implantation Of Trophoblast Cells

??Objective In recent years,the incidence of female infertility has increased to 8-17%.The studies related to discover the indicators for early diagnosis and treatment of infertility to provide the opportunities for improving infertility treatment.Successful implantation requires simultaneous development of the embryo and the endometrium.The embryo undergoes final development,positioning,attachment,and begins to invade the endometrial epithelium in the uterine cavity.Embryo consists of inner cell mass and outer trophoblast cells.The trophoblast cells play a "driver" role in this process.The transformation of cytotrophoblast cells into mesenchymal-like villous trophoblasts is important to successful embryo implantation,and inadequate transformation can lead to abortion,preeclampsia and fetal growth restriction.Many factors affect the process of epithelial-mesenchymal transition(Epithelial-Mesenchymal Transition,EMT)in embryonic trophoblast cells,such as cytokines,chemokines,steroid hormones,and proteases.Epiregulin is a member of the epidermal growth factor family and plays an important role in the reproduction.For example,epiregulin in ovarian granulosa cells promotes oocyte development,and mice lacking epiregulin exhibites delayed oocyte maturation.Epiregulin can also be detected at the placenta and blastocyst implantation sites in the uterus.However,the relationship between epiregulin and EMT process,as well as abortion of embryonic trophoblast cel s has not been reported.Protein glycosylation is the main way of post-translational modification of protein,and plays an important role in many physiological and pathological processes.There are two typical forms of fucosylation: N-fucosylation and O-fucosylation.Protein O-fucosyltransferases 1 and 2(protein O-fucosyltransferase 1/2,po FUT1/po FUT2)are the key enzymes that synthesize O-fucosylation on specific glycoproteins.po FUT1 acts on a substrate containing an epidermal growth factor(epidermal growth factor,EGF)-like domain C2XXXX(S/T)C3 and links the donor L-Fucose to a serine or threonine residue of the protein.po FUT2 transfers fucose to serine or threonine residues of consensus sequences C1XX(S/T)C2 and C2XX(S/T)C3 in thrombospondin type 1 repeat(TSR).Studies have found that mice knocked out of po FUT1 can cause embryonic malformations and even embr yo death.It has been reported that protein O-fucosylation is critical in reproduction.For example,abnormal protein O-fucosylation can inhibits embryonic proliferation and adhesion.po FUT1 can promotes decidualization of uterine stromal cells.In this study,we explored the role and molecular mechanisms of po FUT1 and O-fucosylation in the process of EMT in embryonic trophoblast cel s.The combination of urokinase-type plasminogen activator(urokinase-type plasminogen activator,u PA)O-fucosylated glycoproteins,contains an O-fucosylation modification site on Thr18.And this glycosylation site is located in the binding region of u PA and u PAR.u PA and u PAR can activate plasminogen,degrade the extracellular matrix,and promote cell migration and invasion.u PA / u PAR has been reported in many tumors,and it is closely related to the ability of tumor cells to migrate and invade,but its role in reproduction is less studied.The experiments further explored that po FUT1 alters O-fucosylation on u PA,which affects the ability of u PA to bind with u PAR and trophoblast cel s to migrate and invade.In this study,by using the human villi tissues,human trophoblast cells and pregnant mice model to analyz the functions of epiregulin and po FUT1 in the trophoblast EMT process,the regulatory mechanism of epiregulin on po FUT1 and the effect of O-fucosylated u PA.To explore the mechanism of fucosylation in reproduction,and provide experimental analysis indicators for clinical detection of abortion,and provide an experimental basis for treatment in infertility.?.Methods 1.Analysis the expression of epiregulin and po FUT1 in serum and villi tissues of pregnant women and abortion patients(1)ELISA and Western blot was used to analyze the expression levels of epiregulin,po FUT1 and u PA in the serum of non-pregnant,early pregnant and aborted women.Correlation analysis of epiregulin and po FUT1 in serum of early pregnant and aborted women,and ROC curve analysis of epiregulin and po FUT1;(2)Western blot and Immunofluorescence to detect the expression of epiregulin and po FUT1 in the villous tissues of early pregnant and aborted women,and the expression of EMT markers(E-cadherin/N-cadherin)in the villous tissues of early pregnant and aborted women.2.Epiregulin promotes trophoblast epithelial–mesenchymal transition through po FUT1 and O-fucosylation by po FUT1 on u PA(1)Real-time PCR and Western blot to detect the effects of epiregulin on EMT markers.After treatment with epiregulin,the ability of degrade the matrix by gelatin zymography,and the ability of migration and invasion by transwell;(2)Western blot and immunofluorescence were used to detect the epiregulin through the transcription factor c-Jun/c-Fos affect the expression of po FUT1.Western blot was used to detect the effects of transfection with po FUT1 si RNA and po FUT1 c DNA on po FUT1 expression;(3)Immunoprecipitation was used to detect the transfection of po FUT1 si RNA and po FUT1 c DNA will affect the change of O-Fucose expression on u PA and he binding ability of u P A and u PAR,while the change of O-Fucose expression on u PA and he binding ability of u PA and u PAR after transfection with u PA MUT;(4)Western blot was used to detect the transfection of po FUT1 si RNA and po FUT1 c DNA will affect the downstream signal pathway PI3K/Akt signal pathway,and the effect of u PA MUT transfection on the downstream signal pathway PI3K/Akt signal pathway;(5)Treatment with epiregulin,transfection with po FUT1 si RNA and po FUT1 c DNA,and treatment with TIIA and LY294002 inhibitor.The above experimental group detected the effects of different treatments on the downstream signal pathway PI3K/Akt signal pathway by Western blot.The ability of extracellular matrix degradation by gelatin zymography and invasive ability by Transwell assay.3.Epiregulin promotes embryo implantation in mice(1)After the mouse embryo treatment with an epiregulin-containing medium in vitro,the effect on the expression of po FUT1 in the mouse embryo was detected by immunofluorescence.After the mouse embryos transfection with po FUT1 si RNA and po FUT1 c DN A in vitro,the expression of po FUT1 in mouse embryos was detected by immunofluorescence;(2)After the mouse embryos treatment with epiregulin-containing medium in vitro,the expression of EMT markers and the expression of HLA-G were detected by immunofluorescence;(3)Using a pseudo-pregnant mouse model,observe the implantation of mouse embroys.?.Results 1.Analysis the expression of epiregulin and po FUT1 in serum and villi tissues of pregnant women and abortion patients(1)The expressions of epiregulin,po FUT1 and u PA in the serum of pregnant women were higher than non-pregnant women.The expression of epiregulin,po FUT1 and u PA in the serum of abortion were lower than pregnant women;(2)The expressions of epiregulin and po FUT1 in the villous tissues of early pregnant women were higher than abortion.The expression of N-cadherin in the villous tissues of early pregnant women was higher than abortion,while the expression of E-cadherin in the villous tissues of early pregnant women was lower than abortion.2.Epiregulin promotes trophoblast epithelial–mesenchymal transition through po FUT1 and O-fucosylation by po FUT1 on u PA(1)Epiregulin promoted EMT;Epiregulin promoted the degradation of extracellular matrix and the invasion ability of cells;(2)Epiregulin affected the expression of po FUT1 by activating the transcription factor c-Jun/c-Fos;(3)Transfection with po FUT1 si RN A decreased the expression of po FUT1 and expression of O-Fucose,while expression of po FUT1 and expression of O-Fucose increased after transfection with po FUT1 c DNA.Transfection with po FUT1 si RNA decreased expression of O-Fucose on u PA and decreased the binding capacity of u PA to u PAR,while increased expression of O-Fucose on u PA and the binding capacity after transfected with po FUT1 c DNA;(4)Down-regulated po FUT1 expression by si RN A inhibited PI3K/Akt signaling pathway and decreased degradation of extracellular matrix,while up-regulated po FUT1 by c DNA activates PI3K/Akt signaling pathway and promoted degradation of extracellular matrix;(5)Epiregulin activated PI3K/Akt signaling pathways and promoted EMT behavior of trophoblast cells;transfection with po FUT1 si RNA inhibited PI3K/Akt signaling pathways and impeded EMT behavior of trophoblast cells;treatment with TIIA(AP-1 inhibitor)and LY294002(PI3K inhibitor)inhibited the PI3K/Akt signaling pathway and the occurrence of EMT in cel s was weakened.3.Epiregulin promotes embryo implantation in mice(1)Mouse embryos treatment with epiregulin in medium increased expression of po FUT1.The expression of po FUT1 decreased after transfection with po FUT1 si RNA in vitro with mouse embryos,and the expression of po FUT1 increased after transfection with po FUT1 c DNA;(2)Mouse embryos treatment with epiregulin increased the expression of N-cadherin in,whereas the expression of E-cadherin was decreased.Mouse embryos treatment with epiregulin increased the expression of HLA-G and promoted the ability of migration and invasion in trophoblast cel s;(3)Epiregulin antibody can significantly inhibit the implantation rate of mouse embryos.Transfection with po FUT1 si RN A can also inhibited the implantation rate of mouse embryos.?.Conclusions 1.Analysis the expression of epiregulin and po FUT1 in serum and villi tissues of pregnant women and abortion patients(1)Level of epiregulin and po FUT1 is lower in serum and villi tissues of abortion;(2)Epiregulin and po FUT1 may be suggested as the potential diagnostic biomarkers.2.Epiregulin promotes trophoblast epithelial–mesenc-hymal transition through po FUT1 and O-fucosylation by po FUT1 on u PA(1)Epiregulin promotes EMT in trophoblast cel s;(2)O-fucosylation on u PA will affect the binding ability of u PA and u PAR,and also affect the PI3K/Akt signaling pathway;(3)Epiregulin promotes trophoblast EMT through O-fucosylation on u PA catalysed by po FUT1.3.Epiregulin promotes embryo implantation in mice(1)Epiregulin up-regulates the expression of po FUT1 in mouse embryos;(2)Epiregulin promotes the occurrence of EMT in mouse embryos and has the ability to invade;(3)Epiregulin antibody blocking and knockdown po FUT1 inhibit mouse embryo implantation.