Mechanism Sdudy Of The Regulation Of Protein O-fucosylation On Endometrium Decidualization

Background and Objectives:Endometrial decidualization is a special tissue formed by proliferation and differentiation of uterine interstitial stimulated by decidualization inducing factors.It refers to the process of interstitial remodeling after ovulation due to the change of hormone level,including endometrium stromal cells into decidual cells.Endometrial decidualization is very important for the establishment and maintenance of pregnancy.Protein glycosylation is an important post-translational processing and modification of proteins.It is divided into N-glycosylation and O-glycosylation and is involved in many physiological and pathological processes,including the embryo implantation process.Implant sugar biological studies have shown that: protein glycosylation and endometrial receptivity and other reproductive functions.Fucosylation is an important form of protein glycosylation and is important for endometrial function.However,the molecular mechanism of the role of O-fucosylation catalyzed by the specific protein O-fucosyltransferases in endometrial decidualization is not yet known.This paper aims to investigate the effect of protein O-fucosylation on endometrial decidualization and its mechanism of action.Methods:1.Immunohistochemistry was used to detect the expression of po FUT1 in normal and nonpregnant mice,normal pregnant mice and normal early pregnancy(proliferative / secretory phase)and normal early pregnancy.2.Administration of 10 n M estradiol(E2),1 μM Medroxyprogesterone 17-acetate(MPA),and 0.5 m M dibutyryl c AMP(dbc AMP)to the uterus endometrial stromal cells were induced to undergo decidualization.The expression of po FUT1 gene and protein during endometrial stromal cell decidualization was detected by q RT-PCR,ELISA,Western blot and immunofluorescence staining.3.The effect of po FUT1 on the decidualization of endometrial stromal cells was detected by Western blot and immunofluorescence staining.4.The effect of po FUT1 on the proliferation of endometrial stromal cells during decidualization was detected by Western blot.5.To investigate whether po FUT1 can affect the O-fucosylation of Notch and the effect of Notch’s O-fucosylation on Notch pathway by Western blot and immunoprecipitation.And through the Notch signaling pathway further study po FUT1 regulation of endometrial decidualization.6.In vivo,we injected po FUT1-si RNA into uterine horn of pregnant mice to further verify the role of po FUT1 in mouse endometrial decidualization by immunohistochemistry.Results:1.Collection of normal and non-pregnant mice,uterus and clinical samples of pregnant mice,including normal non-pregnancy(proliferative / secretory phase)and normal early pregnancy tissue,immunohistochemical analysis showed that: The expression of po FUT1 in mouse endometrium was increased with the increase of gestational days in normal pregnant mice.The expression of po FUT1 in proliferative phase,secretory phase and early pregnancy in human endometrium increase in turn.2.Endometrial stromal cells in the process of induction increased expression of po FUT1.Endometrial cells were treated with 10 n M estradiol(E2),1 μM Medroxyprogesterone 17-acetate(MPA),and 0.5 m M dibutyryl c AMP(dbc AMP)Stromal cells were induced to undergo decidualization.Morphological analysis,q RT-PCR,ELISA,Western blot and immunofluorescence staining showed that endometrial stromal cells changed from long spindle to round.The elevated levels of the markers PRL and IGFBP1 prove that endometrial stromal cells have been transformed into decidual cells.Endometrial stromal cells were decidualized and subjected to q RT-PCR,Western blot and immunofluorescence staining.The results showed that the expression of po FUT1 increased after decidualization of endometrial stromal cells compared with the control group.3.po FUT1 promotes the decidualization of endometrial stromal cells.Western blot results showed that compared with the control group,the expression levels of PRL and IGFBP1 were decreased when po FUT1-si RNA was transfected.When transfected with po FUT1-c DNA,endometrial decidualization of the markers PRL and IGFBP1 expression levels were significantly increased.Cell immunofluorescence staining experiment results consistent.4.po FUT1 promotes the proliferation of endometrial stromal cells by activating cyclin.The results of Western blot showed that the expression of PCNA,cyclin D1 CDK2,CDK6 decreased when transfected with po FUT1-si RNA compared with the control group.After po FUT1-c DNA transfection,the expression of PCNA and cyclin in cells increased.5.po FUT1 promotes endometrial decidualization by up-regulating O-fucosylation on Notch and activating the Notch signaling pathway.IP results showed that,compared with the control group,after transfection of pofut1-sirna,IP was carried out with Notch1 antibody,and the O-fucose expression of Notch was down-regulated.And transfected with po FUT1-c DNA,O-Fucose expression was up-regulated.But there was no obvious change in O-fucose on Notch after transfected with po FUT1-mutant compared with the control group.The results of Western blot showed that the expression of Notch signaling molecules DLL-1 and Jagged-1 was down-regulated after transfection with po FUT1-si RNA.After transfection with po FUT1-c DNA,the expression of Notch signaling molecules DLL-1 and Jagged-1 were up-regulated.After transfection with po FUT1-mutant,no significant changes were observed in the signaling pathway.It is demonstrated that O-Fucose expression on Notch has an impact on Notch pathway.At the same time,Western blot results showed that,when the Notch signaling pathway inhibitor was added,the endometrium decidualization process was inhibited,and the expression of the decidualized marker PRL and IGFBP1 was inhibited.6.In vivo experiments in mice further evidence: After injection of pofut1-si RNA in mouse uterine angle,the endometrial decidualization of mice was suppressed,and the embryo implantation rate decreased.The results of immunohistochemistry showed that the expression of endometrial cells IGFBP1,PCNA and Notch signaling molecule DLL-1 were significantly decreased after po FUT1-si RNA injection into the uterine horn of mice.Conclusion:1.Expression of po FUT1 in human endometrium and mouse endometrium showed that the expression level of po FUT1 increased with the increase of gestational days in mouse endometrium.The expression of po FUT1 in proliferative phase,secretory phase,po FUT1 expression in early pregnancy increased in turn.2.Endometrial stromal cells during decidualization of po FUT1 gene and protein expression levels increased.3.po FUT1 promotes proliferation of endometrial stromal cells.4.po FUT1 activates the Notch signaling pathway to promote the endometrial decidualization through the up-regulation of the O-fucosylation of Notch.