ATF3 Facilitates Decidual Prolactin Expression In Human Endometrial Stromal Cells By Increasing FOXO1 Expression

Object:To explore the specific mechanisms of Activating Transcriptional Factor 3(ATF3)in the regulation of human endometrial decidualization.Methods:Immunohistochemical staining was demonstrated to probe the location and expression in endometria.Western blotting was used to investigate ATF3 expression in human endometria of successful conceived women with once embryo transplantation and repeated implantation failure patients.The expression of ATF3 was analyzed by Western blotting and Q-PCR,meanwhile,the expression of miR-135b was detected by Q-PCR during hESCs decidualization induced by 8-Br-cAMP and MPA.Human endometrial stromal cells were infected with Ad-CTL or Ad-ATF3,otherwise stimulated with 8-Br-cAMP and MPA after knocking down ATF3 or being transfected with miR-135b,all treated hESCs were applied for 3 days and 6 days.Q-PCR and ELFA was performed to measure expression of decidual prolactin,and immunofluorescent assay was applied to inspect hESCs' morphology.ChIP and Q-PCR was performed to examine the interaction between ATF3 and miR-135b.The expression of FOXO1,regulated by ATF3 and miR-135b,was detected by Western blotting and Q-PCR.Results:ATF3 was specifically expressed in stromal cells and epithelial cells in human endometria and was aberrantly decreased expressed in endometrial samples of recurrent implantation failure patients(n=31)compared with successful fertilized women with once embryo transplantation(n=29)(P<0.001).When stimulated with both 8-Br-cAMP and MPA,the ATF3 mRNA level in hESCs was increased by 5-fold in 1 h and its protein expression was increased by 4-fold after 16 h-Adenovirus-mediated overexpression of ATF3 in hESCs markedly increased dPRL mRNA expression(P<0.01)in a concentration-dependent manner,simultaneously making hESCs demonstrate a morphological transformation from fibroblast-like to decidual cell-like.On the contrary,knocking down ATF3 partially reversed the increase in dPRL secretion(P<0.01)induced by 8-Br-cAMP and MPA.MiR-135b was decreased to 30%after 16 hours' decidualization in hESCs.MiR-135b directly targeted FOXO1 3'UTR and suppressed its protein expression,subsequently decreased dPRL secretion.ATF3 depressed miR-135b activity by specifically binding to its promoter region increasing FOXO1 expression,and consequently facilitating dPRL secretion during decidualization(P<0.001).Conclusion:This study first illustrates that ATF3 promoted FOXO1 expression by transcriptionally inhibiting miR-135b,consequently facilitating decidual prolactin expression in hESCs during decidualization induced by 8-Br-cAMP and MPA in vitro.by increasing FOXO1 expression...