O-GlcNAc Influences Modification On Endometrial Receptivity By Promoting Endometrial Cell Proliferation,Migration And Invasion

Ⅰ.Objective O-Glc NAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine(Glc NAc)molecule to the hydroxyl group of Ser/Thr residues in cytoplasmic and nuclear proteins by the enzyme O-linked-Nacetylglucosamine transferase(OGT),whereas the enzyme O-Glc NAcase(OGA)removes the sugar moiety from proteins.Endometrial receptivity refers to a limited time period when the endometrium is conducive to blastocyst growth,attachment and the subsequent events of implantation.The duration of this state is called the ‘window of implantation’.At present,the study of O-Glc NAc modification is mainly focused on the occurrence and metastasis of tumor.The behavior of recognition and adhesion of the embryo to the endometrium is strikingly similar to that of invasive cancer cells.Glycosylation of proteins plays an important role in embryonic development,yet the role of O-Glc NAcylationin the regulation of implantation is just now being defined.In this study,the differences expression of O-Glc NAcylation protein in endometrium was detected in mice and human at different stages.Endometrial carcinoma cell lines RL95-2 and HEC-1A were used to simulate the receptive epithelial cells and nonreceptive epithelial cells respectively to analyze the expression of O-Glc NAcylation.Using RNAi technology to regulate the expression of OGT and OGA,the effects of O-Glc NAcylation on the proliferation,migration and invasion of endometrium cells were analyzed,and the regulatory effects on EMT related proteins of endometrium cells were detected.The chorionic tumor cell line JAR was used to simulate invasive embryo,to detect the effect of OGT expression in endometrial cells on the adhesion between JAR cells and endometrial cells.We revealed the possible correlation and molecular mechanism of O-Glc NAcylation to regulate embryo implantation and endometrium receptivity.Therefore,it provides a theoretical basis for broadening the glycosylation modification of proteins in clinical reproduction.Ⅱ.Methods1.Immunohistochemistry was used to detect the expression and distribution of O-Glc NAc modified protein in pregnant mice endometrium during the perimplantation period(D1-D5).2.Immunohistochemistry and western blot were used to detect the expression and localization of O-Glc NAc modified protein in endometrium of different menstrual cycle.3.Real-time PCR was performed to detect the changes of OGT expression in the endometrium at different stages of the menstrual cycle.4.Endometrial carcinoma cell lines RL95-2 and HEC-1A were used to simulate the receptive epithelial cells and nonreceptive epithelial cells respectively.Detection of ogt and oga gene expression in RL95-2 and HEC-1A cells by RT-q PCR.5.In RL95-2 cells with high expression of OGT,an ogt small interference RNA fragment was transfected into cells.On the other hand,treatment of HEC-1A with oga si RNA and detected by RT-q PCR and western blot.6.A chorionic tumor cell line JAR was used to simulate invasive embryos.The effect of O-Glc NAcylation on the adhesion of endometrial cells to embryonic cells was studied by adhesion assay.7.Cell Counting Kit-8 assay analyse the effect on cell proliferation ability.The invasion and migration abilities were evaluated using transwell assay and would healing assay.8.Western blot was used to detect the effect of O-Glc NAcylation on the expression of EMT related proteins.Ⅲ.Results1.The level of endometrial O-Glc NAc modification increased gradually on D1-D4 and reached its peak on D4,then decreased after D5.O-Glc NAc modification could be detected in all the tissues of the endometrium in perimplantation period of the mice,but mainly expressed in the stromal cells.2.The expression of O-Glc NAc modified protein in human secretory endometrium was higher than that in proliferative phase.O-Glc NAc modified protein was hardly expressed in endometrium during proliferative phase.But during the secretory phase,the expression level increased significantly.In human,O-Glc NAc modified protein mainly expressed in gland epithelium and luminal epithelium.3.The OGT levels in secretory endometrium was higher than proliferative phase.4.The expression of ogt gene in RL95-2 was significantly higher than that in HEC-1A.The expression of oga gene in RL95-2 was significantly lower than that in HEC-1A.5.The level of OGT m RNA and protein and O-Glc NAcylation decreased significantly after OGT si RNA was transfected into RL95-2 cells.OGA si RNA was transfected into HEC-1A cells also significantly attenuated the increased expression of OGA protein and m RNA,meanwhile decreased the O-Glc NAc modification levels.6.The increase of O-Glc NAc modification level will enhance endometrial cell proliferation,migration and invasion ability.7.The increase of O-Glc NAcylation level will affect the expression of EMT related proteins,promote the occurrence of EMT process,and thereby affect the migration and invasion ability of cells.8.The adhesion ability of JAR to RL95-2 was stronger than that of HEC-1A.The increase of O-Glc NAc modification level will increase cell adhesion rate.Ⅳ.Conclusions1.The O-Glc NAcylation in implantation endometrium was higher than that in non-implantation.It may play a positive role in regulating the endometrial receptivity.2.The O-Glc NAc modification level enhanced endometrial cells proliferation,migration and invasion ability,and promoted cell adhesion rate between endometrial cells and embryonic cells,thus contributing to the establishment of endometrial receptivity and embryo implantation.3.The O-Glc NAcylation level may affect the migration and invasion ability of cells by affecting the expression of EMT related proteins.