ZMYND8 Facilitates PGR Protein Stability And FOXO1 Expression During Decidualization Of Human Endometrial Stromal Cell

ObjectiveRecurrent implantation failure(RIF)is a difficult problem in assisted reproductive technology,which is the core technology to solve infertility.The occurrence of RIF is influenced by uterine receptivity,and the successful establishment of uterine receptivity is regulated by steroid hormones,and the ZMYND8 gene has been reported to be closely related to steroid hormones in the literature.The ZMYND8 gene encodes the activated protein kinase C receptor 7 protein as a transcription factor involved in the transcriptional regulation of multiple genes.Therefore,this experiment needs to clarify the relationship between RIF causing infertility and ZMYND8 expression levels.The steroid hormone progesterone(P4)plays an important role in the decidualization of endometrial stromal cells.Because ZMYND8 gene is closely related to steroid hormones,we speculate that ZMYND8 gene may be involved in the process of decidualization regulated by P4.At present,the role of ZMYND8 in the process of decidualization is not clear.This study was designed to investigate the role of ZMYND8 in the process of decidualization and its regulationMethods1,Endometrial tissues were collected from 10 patients with RIF combined with infertility and 8 normal fertile women at mid-secretory stage,and expression level of ZMYND8 protein was detected by Western Blot.Endometrial tissues were collected from fertile women at different periods of the menstrual cycle,and expression levels of the ZMYND8 mRNA were detected by quantitative real-time PCR(qRT-PCR),and ZMYND8 protein expression localization was detected in the tissues by immunohistochemical staining.Human primary endometrial stromal cells were treated with in vitro induced decidualization for 0,48,96 and 144 h.The expression of ZMYND8,the decidual marker molecules prolactin(PRL)and insulin-like growth factor binding protein 1(IGFBP1)mRNA and or protein were detected by qRT-PCR and Western Blot.Human immortalized endometrial stromal cells were treated with MPA and c AMP for 0,48,96 and 144 h for in vitro induced decidualization.The expression of ZMYND8,the decidual marker molecules PRL and forkhead box protein O1(FOXO1)mRNA and or protein were detected by qRT-PCR and Western Blot.2,The levels of RPL mRNA,IGFBP1 protein and ZMYND8 mRNA and protein were detected by qRT-PCR and Western Blot in human immortalized endometrial stromal cells after si RNA knockdown of ZMYND8 expression induced decidualization treatment for 96 h;F-actin staining was performed to detect the changes of cell morphology in human immortalized endometrial stromal cells after si RNA knockdown of ZMYND8 expression in decidualization.Fresh human endometrial tissues were collected for in vitro isolation and culture of primary stromal cells.qRT-PCR was performed to detect the levels of RPL and IGFBP1 mRNA;F-actin staining was performed to detect the changes of cell morphology in the decidualization of human primary endometrial stromal cells after si RNA knockdown of ZMYND8 expression.After si RNA knockdown of ZMYND8 expression in human immortalized endometrial stromal cells,Ki67 fluorescence staining and MTT assay were performed to detect cell proliferation and qRT-PCR to detect the level of ki67 mRNA.3,Human immortalized endometrial stromal cells were induced to decidualization using different reagents.qRT-PCR was performed to detect changes in the levels of ZMYND8 mRNA.The expression of ZMYND8 mRNA was detected by qRT-PCR using different reagents to induce decidualization with RU486,a progesterone receptor inhibitor.Human immortalized endometrial stromal cells were transfected with plasmid to overexpress PGR followed by decidual treatment to collect 96 h protein,and Western Blot to detect ZMYND8 protein expression level;96h mRNA and protein were collected in immortalized endometrial stromal cells after knocking down ZMYND8 followed by decidual treatment,and qRT-PCR and Western Blot to detect PGR expression Altered.The changes in PGR protein levels were detected by Western Blot after knockdown of ZMYND8 expression in immortalized endometrial stromal cells treated with induction of decidualization for 96 h followed by addition of the protein synthesis inhibitor CHX;or add the proteasome inhibitor MG-132 and Western Blot to detect changes in PGR protein levels.4,After ZMYND8 knockdown and induction of decidualization in human immortalized endometrial stromal cells,FOXO1 mRNA was detected by qRTPCR and FOXO1 protein expression was detected by Western Blot.Human immortalized endometrial stromal cells were knocked down by ZMYND8 followed by decidualization treatment for 96 h.Western Blot was performed to detect the protein expression levels of AKT and p-AKT(Ser 473),the upstream molecules of FOXO1,and p-FOXO1(Ser 256),GSK-3β,and GSK-3β(Ser 9)of the AKT signaling pathway.Human immortalized endometrial stromal cells were knocked down by ZMYND8 while overexpressing PGR before inducing decidual,and the protein expression levels of p-FOXO1(Ser 256),GSK-3β,and GSK-3β(Ser 9)of AKT signaling pathway were detected by Western Blot.Human immortalized endometrial stromal cells were knocked down by ZMYND8 followed by AKT inhibitor(MK2206)treatment and then decidualization for 96 h.qRT-PCR was performed to detect PRL,FOXO1,CCNB1,CCNB2 mRNA expression levels.Results1,ZMYND8 protein levels were significantly lower on endometrium at middle secretory phases in RIF patients than in normal fertile women.The expression of ZMYND8 in tissues was lowest on the stroma of of endometrium of fertile women during the estrogen-dominant proliferative phase of the mensturl cycle,and gradually increased on the stroma during the entry into the progestin-dominant secretory phase,with high expression in the stroma during the middle and late secretory phases.The expression of decidual marker molecules PRL,IGFBP1 mRNA and IGFBP1 protein increased gradually and steadily,and ZMYND8 mRNA and protein expression increased gradually and steadily after in vitro induction of decidualization of human primary endometrial stromal cells.Moreover,the expression of decidual marker molecules PRL mRNA and FOXO1 protein was also gradually and steadily increased after in vitro induction of human immortalized endometrial stromal cells decidualization.The human ZMYND8 protein was mainly localized on the nucleus in immortalized endometrial stromal cells,and the expression was increased after induction of decidualization by immunofluorescence staining.2,Expression level of ZMYND8 mRNA and protein were both significantly reduced in human immortalized endometrial stromal cells after ZMYND8 knockdown,which was also accompanied by a significant reduction in RPL mRNA and IGFBP1 protein levels.The decidualization of imortalized human endometrial stromal cells morphological transformation were significantly inhibited with ZMYND8 knockdown.The expression levels of RPL and IGFBP1 mRNA were significantly reduced after ZMYND8 knockdown on human primary endometrial stromal cells.Cellular decidualization morphological transformation was significantly inhibited after ZMYND8 knockdown in human primary endometrial stromal cells.Cell proliferation was significantly inhibited and Ki67 mRNA levels were reduced after ZMYND8 knockdown in human immortalized endometrial stromal cells.3,ZMYND8 mRNA levels were not altered during c AMP-induced decidualization of human immortalized endometrial stromal cells,whereas MPA could significantly increase ZMYND8 mRNA expression,and ZMYND8 mRNA levels were the highest when c AMP and MPA were combined.The addition of RU486 was able to significantly reduce the MPA-induced and MPA-c AMPcoupled-induced ZMYND8 mRNA levels during the decidualization.Human immortalized endometrial stromal cells transfected with plasmids overexpressing PGR showed a significant decrease in ZMYND8 protein levels,and double immunostaining fluorescence experiments revealed that ZMYND8co-localized with PGR fluorescence.PGR protein levels were significantly reduced after ZMYND8 knockdown in human immortalized endometrial stromal cells,but PGR mRNA levels were not significantly altered.Protein degradation assay revealed that PGR protein was degraded earlier in the group with reduced ZMYND8 expression than in the control group.In contrast,the proteasome inhibitor MG-132 can effectively inhibit the rapid degradation of PGR protein.4,Reduced ZMYND8 expression significantly decreased FOXO1 mRNA and protein levels.The expression of p-AKT(Ser 473)protein,a molecule upstream of FOXO1,and p-FOXO1(Ser 256)and GSK-3β(Ser 9)protein of the AKT signaling pathway were significantly increased after ZMYND8 knockdown in human immortalized endometrial stromal cells.The p-FOXO1(Ser 256)and GSK-3β(Ser 9)protein expression of the AKT signaling pathway was significantly rescued and the protein level expression of FOXO1 was rescued and increased after knockdown of ZMYND8 in human immortalized endometrial stromal cells with concomitant overexpression of PGR and then induction of decidualization.IGFBP1,FOXO1,CCNB1,and CCNB2 mRNA levels were rescued after ZMYND8 knockdown followed by MK2206 treatment in human immortalized endometrial stromal cells.Conclusions1,Reduced fertility in RIF patients may be associated with decreased ZMYND8 expression.The upregulation of ZMYND8 expression is a prominent feature of endometrial stromal cells undergoing decidualization.ZMYND8 may be involved in maintaining decidualization.2,The decreased expression of ZMYND8 resulted in impaired decidualization and proliferation of human endometrial stromal cell.3,The expression of ZMYND8 is mainly regulated by P4 in decidualization of human endometrial stromal cell.Reduced expression of ZMYND8 affects the protein stability of PGR,mainly through the proteasomal degradation pathway.4,Reduced expression of ZMYND8 in human immortalized endometrial stromal cells leads to impaired decidualization via the P4-PGR-AKT-FOXO1 axis.