The Role Of Histone Methyltransferase In Impaired Endometrial Decidualization Induced By BPA

Objective BPA is a widespread environmental endocrine disruptor,which is related to female reproductive endocrine diseases.Studies have found that BPA exposure can reduce the number of embryo implantation in mice and affect the expression of decidualization markers.However,the molecular mechanism of BPA exposure on abnormal endometrial receptivity has not been clarified.Endometrium decidualization is periodically regulated by steroid hormones.Hormone regulate histone modifications in endometrial stromal cells to activate or inhibite of genes transcription during decidualization.Histone H3K4me3 and H3K27me3 modifications can jointly play a role in the promoter region of target genes.They are functionally balanced to maintain gene homeostasis.Histone methyltransferase MLL1 mediates the modification of H3K4me3 and plays a role in gene transcription,the function in endometrial decidualization is unknown.EZH2 mediates H3K27me3,and inhibits genes transcription,the decreased expression of EZH2 has been shown involved in endometrial decidualization.Histone modifications are susceptible to external environmental factors or hormone levels.Whether BPA exposure affects histone modification during endometrial decidualization has not been reported.This part will study whether histone modifications are involved in the process of BPA affecting endometrial stromal cells decidualization.Methods 1)Human endometrial stromal cells were treated with medroxyprogesterone acetate(MPA)and dibutyl cyclic adenosine monophosphate(db-c AMP)to induce in vitro decidualization,different concentrations of BPA(0p M,10 p M,100 p M,1n M,10 n M,100 n M,1 m,10μM)was also added to the cultured cells,the morphological changes of endometrial stromal cells were observed.Western Blot and RT-q PCR were used to detect the expression of HOXA10,PRL,IGFBP-1 and the expression of histone methylated transferase MLL1 and EZH2 protein and m RNA expression.2)Cellular immunofluorescence was used to detect the total H3K4me3 and H3K27me3 proteins expression after different concentrations of BPA(0p M,10 p M,10 n M,10m)treatment during decidualization.3)The expression levels of H3K4me3 and H3K27me3 at promoter regions of decidual key genes PRL,IGFBP-1,HOXA10 were detected by CHIP-q PCR.4)While BPA was treated,estrogen receptor antagonist(ICI 182780)or control solvent dimethyl sulfoxide(DMSO)was added,Western blot was used to detect the protein expression of MLL1,EZH2 and HOXA10.Results 1)During in vitro decidualization,endometrial stromal cells changed their morphology from long spindle to blunt round,epithelioid cells.After BPA of 10 n M and10μM treatment,the cells did not show obvious decidualization changes,but remained long spindle,non-decidualized morphology.During decidualization,the expression of MLL1 gradually increased as the BPA concentration increased.The activity of MLL1 decreased significantly.BPA exposure also increased the expression of EZH2,the expressions of HOXA10,PRL and IGFBP-1 were down-regulated after BPA treatment.2)when BPA concentrations were 10 n M and 10μM,the fluorescence intensity of total H3K4me3 were decreased.However,the fluorescence intensity of the total H3K27me3 protein increased.3)BPA exposure during decidualization decreased the level of H3K4me3 modification at promoter regions of HOXA10,PRL and IGFBP-1,while the level of H3K27me3 increased.4)BPA treatment decreased the protein expression of MLL1 and HOXA10 in endometrial stromal cells and increased EZH2 expression,while the use of estrogen receptor antagonists ICI 182,780 reversed the effects of BPA on MLL1,EZH2 and HOXA10.Conclusion BPA exposure inhibited MLL1-mediated H3K4me3 modification and increased EZH2-mediated H3K27me3 modification,so that gene transcription tended to be inhibited and the expression of decidual related genes were reduced.Objective Repeated transplantation failure has become a knotty problem that inhibited pregnancy rate in assisted reproductive technology.Endometrial decidualization is a key speed-limiting step in the process of embryo implantation.A large number of regulatory molecules and gene expression changes participate in this precise regulation process.To study of the mechanism of endometrial decidualization is particularly important.The first part of the study found that BPA can regulate the expression of histone methylated transferase MLL1 and EZH2,thus affecting decidual marker genes expression,and shows the important role of two histone modifications during decidualization.Previous studies have confirmed that EZH2 mediated H3K27me3 modification participates in the process of decidualization.But the role of another histone methylated transferase MLL1 in decidualization has not been reported.MLL1 mediates histone H3K4me3 modification,which can promote target genes transcription,and MLL1 can regulate the expression of HOXA gene family.HOXA10,HOXA11 are marker genes of endometrial receptivity.MLL1 also has a steroid hormone receptor binding site,which may be related to hormone signal transmission.Whether MLL1 is regulated by steroid hormone signals during decidualization needs further research.In this part,the expression of MLL1 in endometrium tissues and the mechanism in the process of decidualization will be discussed.Methods 1)The proliferative and secretory endometrium tissue from repeated implantation failure(RIF)and normal patients(control group)were collected,and the expression MLL1 and HOXA10 in endometrial tissues were detected by immunohistochemistry and RT-PCR.2)Human primary endometrial stromal cells and the human endometrial stromal cell lines(HESCs)were cultured,and then treated with MPA and c AMP alone or in combination.Detect the protein or m RNA expression of MLL1,HOXA10,PRL,IGFBP-1,H3K4me3.3)MLL1 inhibitor MM-102 was added during decidualization,then detect the HOXA10,PRL,IGFBP-1 m RNA expression.4)After treatment with control si RNA or MLL1 si RNA,followed by decidualization for 0 or 4 days.Expression of PGR and its target genes Hand2,STAT3,FOXO1,HOXA10 m RNA were detected during decidualization.5)co-IP assay was performed to evaluate whether ER and MLL1 were combined during decidualization.6)CHIP-q PCR assay was conducted to detect whether ER and MLL1 were recruited to the promotor region of PGR,and whether MLL1,as a coregulator of ER wasrequiredtoPGRtranscription during decidualization.Results 1)The level of MLL1 was elevated in the secretory endometrium compared to proliferative phase from patients in the control group,while the expression of MLL1 was not significantly changed in the secretory endometrium from patients with RIF.MLL1 in the secretory endometrium of patients with RIF was significantly lower than that of patients with normal pregnancy.The expression trends of HOXA10 and MLL1 were the same.In addition,the secretory endometrium MLL1 and HOXA10 expression were positively correlated.2)After combined treatment of human endometrial stromal cells with decidualization hormone MPA and c AMP for 6 daysin vitro,the expression level of MLL1 increases most significantly,and the level of total H3K4me3 modification in cells also increases.3)MM-102 reduced decidual related genes expression.4)After MLL1 expression was inhibited by si RNA followed by decidualization,the expression of PGR gene is significantly reduced,and the expression of target genes Hand2,STAT3,FOXO1,HOXA10 were also affected.5)During decidualization,the binding of MLL1 to ER proteins were increased,MLL1 may act as co-regulator of ER to activate target genes transcription.6)ER and MLL1 can both be recruited to ERE/Sp1 loci at PGR promoter region,while down-regulated expression of MLL1 reduces the recruitment of ER to ERE/Sp1 loci at PGR promoter region.Conclusion Histone methylation transferase MLL1 can responde to decidual hormone regulation,and it acts as a co-regulator of ER to activate PGR gene transcription activation,thus influence PGR signal transmission during decidualization.MLL1 plays a key role in the process of endometrial stromal cell decidualization and the establishment of endometrial receptivity.