Mutation Of N-linked Glycosylation In Osteopontin Affected The Establishment Of Endometrial Receptivity

??ObjectiveGlycosylation is one of the common post-translational modification of proteins.Glycosylation is one of the common post-translational modification of proteins.These modifications,covalently added by organic and inorganic groups,can occur on almost all known proteins in mammalian cells,and they represent a fine regulatory mechanism.The genetic information of DNA is extended from linear to the panoramic view of three-dimensional structure.The glycosylation can be divided into N-glycosylation,O-glycosylation,C-mannose saccharification and glycosylation anchored by phosphatidylinositol.The glycosylation of proteins can change the spatial structure and stability of peptide chain,and thus affect the biological function of cells,including the transport of glycoproteins,the transmission of cell signals,the recognition and adhesion between cells,and so on.At present,many studies have found that in the epigenetic changes associated with tumor,glycosylation not only directly affects the growth,survival,invasion and metastasis of tumor cells,but also promotes tumor induced immunomodulation and final metastasis.Osteopontin(OPN)is a secretory phosphorylated glycoprotein of the N-binding glycoprotein family.OPN regulates cell adhesion,cell migration,and cell matrix interaction by interacting with integrin receptors on the cell surface.These functions are closely related to the proliferation,migration and invasion of tumor cells,and these functions depend largely on the post-translational modification of proteins.However,the role of OPN N-glycosylation in the reproductive process is unclear.In this study,human endometrial carcinoma cell lines(RL95-2 cells,HEC-1A cells)were selected to simulate the high or low receptive state of endometrium.The OPN-N glycosylation mutant plasmid was constructed and transfected into the two endometrial carcinoma cell lines,and then cloned by CCK-8 assay.The effects of N-glycosylation on the proliferation,migration and invasion of endometrial cells in high and low receptive state were detected by cell scratch assay and Transwell chamber assay.The molecular mechanism of OPN-N glycosylation on the regulation of endometrial receptive state was further studied by western blot experiment.However,the role of OPN N glycosylation in the reproductive process is not clear.In this study,human endometrial cancer cell lines(RL95-2 cells,HEC-1A cells)were selected to simulate the endometrium of high and low acceptance state,and OPN-N glycosylation mutant plasmid was constructed and transfected into these two endometrial cancer cells.The effects of N-glycosylation modification on the proliferation,migration and invasion of endometrial cells in high and low receiving state were detected by CCK-8 assay,colony formation assay,cell scratch assay and Transwell chamber assay.The molecular mechanisms of the regulation of OPN-N glycosylation on endometrial receptor status was further investigated by RT-PCR and western blot.??Methods 1.Select RL95-2 cells and HEC-1A cells to simulate endometrium with high and low acceptance status;2.The OPN-N glycosylation mutant plasmid was constructed and transfected into the above two endometrial cancer cell lines;3.The effects of N-glycosylation modification on protein localization and its properties were determined by immunofluorescence and western blot.4.The effects of N-glycosylation modification on cell proliferation,migration and invasion ability were detected by CCK-8 assay,colony formation assay,cell scratch assay and Transwell chamber assay.5.The molecular mechanism of the regulation of OPN-N glycosylation on endometrial receptor status was further studied by western blot.??Results 1.OPN N glycosylation modification does not affect the localization of OPN in cells;2.After OPN removes N glycosylation modification,OPN core protein is more easily detected;3.OPN N glycosylation modification promotes the proliferation of RL95-2 cells;However,it does not promote the proliferation of HEC-1A cells.4.OPN N glycosylation modification promotes migration and invasion of RL95-2 HEC-1A cells.5.MAPK/ERK and PI3K/Akt pathway involved in OPN N-glycosylation modification regulates migration and invasion of endometrial cells.??Conclusions 1.OPN N glycosylation modification promotes the growth of RL95-2 cells;2.OPN N glycosylation modification promotes migration and invasion of RL95-2 and HEC-1A cells;3.MAPK/ERK and PI3K/Akt pathways are involved in the regulation of OPN N-glycosylation to regulate the migration and invasion of endometrial cells;4.OPN N glycosylation modification by activation of MAPK/ERK involved in transcription factor Snail regulates E-cadherin and Vimentin expression to promote EMT(Epithelial-Mesenchymal Transition,EMT)process in RL95-2 cells and HEC-1A cells;5.OPN N glycosylation modification by activation of PI3K/Akt involved in the transcription factor ?-catenin regulates MMP-2/9 expression promotes the adhesion of RL95-2 cells and HEC-1A cells,thereby regulating its migration and invasion process.