Progesterone Up-regulates The Expression Of PoFUT1 Gene And Promotion Embryonic Cell Proliferation And Adhesion

Background and Objectives:Progesterone plays an important role in embryonic maturation and implantation.Progesterone deficiency may cause embryonic dysplasia,leading to the occurrence of abortion eventually.Progesterone promotes the proliferation and implantation of embryonic cells by regulating the expression of multiple molecules.Protein glycosylation is an important post-translational modification step,which involved in embryonic development and embryo implantation.Protein glycosylation mainly includes N-glycosylation and O-glycosylation.The roles of protein-fucosyltransferases,a protein-catalyzed protein-O-fucosyltransferase,in embryonic development and implantation have not been reported yet.Activation protein-1(AP-1)is a transcription factor involved in a variety of physiological processes,including growth,differentiation,tissue remodeling,migration and invasion.AP-1 is widely expressed and activated in human placenta,which involved in the regulation of placental formation and fetal development.This study was to investigate the mechanism of progesterone in the regulation of O-glycosylation during embryo implantation.Progesterone promoted the phosphorylation of c-Fos and c-Jun,as well as the expression of poFUT1,and the proliferation and adhesion abilities of embryonic cells.Experiments can help to reveal the role and mechanism of 6implantation of glycobiology,and provide new ideas for clinical medicine research and treatment.Methods:1.The expression of progesterone and poFUT1 in the serum of non-pregnant healthy women,early pregnancy women and threatened abortion women were detected by ELISA and Western blot.The expression of poFUT1 in human embryonic villi was detected by immunofluorescence.2.The effects of progesterone,as well as progesterone receptor antagonist mifepristone(RU486)on the expression of poFUT1 in human embryonic cells(JAR),were detected by Real-time PCR,Western blot and immunofluorescence.3.Western blot,EMSA and immunofluorescence were used to investigate the regulation mechanism of progesterone on the expression of poFUT1,and determine that progesterone could activate the transcriptional activity of transcription factor AP-1.4.The transcription factor AP-1 was directly effect the promoter region of poFUT1 and promoted the transcription of poFUT1 which was detected by CHIP.The expression change of poFUT1 after transfected with c-Jun/c-Fos siRNA,or treated with the AP-1 inhibitor(TIIA)was detected with Real-time PCR and Western blot.5.Embryo adhesion rate in 9 groups,including the normal control group,progesterone(10-5mol/L),progesterone(10-5mol/L)and mifepristone(10-5mol/L),transfected with c-Jun/c-Fos siRNA interfering plasmids,using AP-1 inhibitors TIIA and combined with progesterone were assayed in vitro implantation model with the co-cultured endometrial RL95-2/HEC-1A cells and embryonic JAR cells.6.The proliferation of JAR cells was detected by Western blot,immunofluorescence and CCK-8.Results:1.Serum samples from non-pregnant healthy women,early pregnancy women and threatened abortion women were collected.The serum levels of progesterone and poFUT1 in early pregnancy women were higher than those in threatened abortion women and non-pregnant healthy women.Progesterone and poFUT1 levels were 7lower than early pregnancy women.The expression of progesterone in the serum was positively correlated with poFUT1.Immunofluorescence was used to determine the location of poFUT1 in human chorionic villi.2.Progesterone promoted the expression of poFUT1 in JAR cells.The results of Real-time PCR and Western blot showed that progesterone(10-5mol/L)could significantly increase the expression of poFUT1 in JAR cells afer treated with different concentrations of progesterone.The results of Real-time PCR,Western blot and immunofluorescence showed that treated with progesterone and the progesterone receptor antagonist(mifepristone,RU486)could antagonize the effect of progesterone,and downregulated the expression of poFUT1 in JAR cells.The expression of poFUT1 was significantly decreased after transfected with poFUT1 siRNA,but restored after the addition of progesterone.3.Progesterone promotes the expression of poFUT1 and regulates JAR cells' ability of to proliferation and adhesion.Real-time PCR,Western blot and immunofluorescence shows that the combination of progesterone in JAR cells with transfected poFUT1 siRNA can restore the expression of poFUT1.With Western blot,CCK-8 and immunofluorescence and other methods,the results show that progesterone regulates the poFUT1 expression to affect proliferation and adhesion of JAR cells.4.Progesterone activatesd the transcriptional activity of transcription factor AP-1.The results of Western blot,EMSA and immunofluorescence showed that progesterone(10-5mol/L)could significantly increase the expression of transcription factor AP-1(c-Jun/c-Fos)and promote its accumulation in the nucleus,while the antagonist RU486 significantly reduced the activity of transcription factor AP-1.5.AP-1 can regulate the expression of downstream target gene poFUT1 directly.The results of Real-time PCR and Western blot showed that the down-regulated AP-1 transcriptional activity could significantly decrease the expression of poFUT1 which compared with the control group.We further confirmed that AP-1 could act directly in the promoter region of poFUT1,causing the transcription of downstream target gene poFUT1 and affecting its expression level by CHIP.6.Progesterone upregulated the expression of poFUT1 by activating transcription factor AP-1.Progesterone upregulated the expression of poFUT1,and the expression level of poFUT1 could affect the proliferation ability of embryonic cells.Western blot,CCK-8 and immunofluorescence analysis showed that progesterone significantly increased the proliferation of embryonic cells compared with the control group.Compared with the transfected control group,c-Jun/c-Fos siRNA could significantly decrease the proliferative capacity,and progesterone could restore the low proliferative capacity of transfected with c-Jun /c-Fos siRNA and pretreated AP-1 inhibitor TIIA;further studies have shown that progesterone enhanced the expression of poFUT1 to promote the proliferation of embryonic cells.7.The adhesion rate was observed by in vitro implantation model with the co-cultured endometrial RL95-2/HEC-1A cells and embryonic JAR cells.Compared with the control group,progesterone could significantly increase the adhesion rate of the embryonic cells,while c-Jun/c-Fos siRNA could significantly decrease the adhesion rate of embryonic cells compared with the control group.Progesterone could restore the adhesion rate of c-Jun/c-Fos siRNA pretreated low embryonic cells adhesion rate;progesterone was able to restore the embryo low adhesion rate of pretreatment with AP-1 inhibitor TIIA also;Further studies showed that progesterone could significantly recover of poFUT1 siRNA resulted in low embryonic adhesion.Progesterone enhanced the adhesion of embryonic cells to endometrial cells by promoting the expression of poFUT1.Conclusion:1.Serum levels of progesterone and poFUT1 in early pregnancy women are higher than those in threatened abortion women,and progesterone is positively correlated with poFUT1.The expression of poFUT1 is localized in human embryonic villi tissue.2.Progesterone upregulates the expression of poFUT1 in embryonic cells.3.Progesterone activates transcription factor AP-1,and upregulate poFUT1 expression.4.Progesterone promotes the proliferation of embryonic cells.5.Progesterone activates the transcriptional activity of transcription factor AP-1,which regulates the transcription of target gene poFUT1 and promotes the adhesion between embryonic cells and endometrial cells.