Regulaton Of DNA Methylation In Trophoblast Defferentiation

Placenta is a unique and highly dynamic temporary organ formed during pregnancy.DNA methylation plays a role in placental development and trophoblastic fusion.Abnormal DNA methylation may participate in the occurrence of preeclampsia.In this study,utilized the expression pattern of DNA methyltransferases 1,3A,3B in human normal and preeclamptic placentas and elucidate the role of DNMTs in the process of trophoblast fusion.We also screened the differentially expressed genes potentially regulated by DNA methylation during trophoblast fusion.Finally we explored the relationship between DNMTs expression and CREB signaling pathway activation in trophoblast fusion.The results are as follows: 1.Expression pattern and methylation level of DNMTs in trophoblast cellsHuman chorionic villi in early pregnancy,syncytiotrophoblast reconstructed model,human primary trophoblastic(PHT)spontaneous fusion model and BeWo cell induced fusion were used to study the expression of DNMTs in trophoblast cells by immunohistochemistry,immunofluorescence and Western Blot.The results showed that DNMTs and 5mC were highly expressed in the cytotrophoblast compared with the neotrophic syntrophoblast;trophoblast spontaneous fusion and BeWo cell fusion inducted by forskolin is accompanied by DNMTs decreased expression.2.The role of DNMTs in trophoblastic fusionDNMTs expression was changed in BeWo cell line under DNA methyltransferase inhibitor 5-aza treatment and forced expression of DNMTs.It is shown that the rate of BeWo cell fusion is enhanced with DNMTs were inhibition but decreased in cells with DNMTs overexpression.The interrelationship between DNMTs expression and CREB signaling pathways activation.The results of Western Blot showed that H89 could significantly inhibit the level of CREB phosphorylation and expression of syncytiotrophoblastic markers.Down-regulation of DNMTs caused by spontaneous fusion failed to recover after H89 treatment.The expression of DNMTs was decreased and phosphorylated CREB was increased in Bewo cells induction of 5-aza.3.Screening of differentially expressed genes potentially regulated by DNA methylation during trophoblastic fusionBased on the methylation chip sequencing data generated from the primary trophoblast cells spontaneous fusion and cytotrophoblast as well as the date downloaded from the GEO database,GO and KEGG clustering were used to screen genes with the demethylation and differentially upregulated genes.59 genes were demethylated and up-regulated during the trophoblastic fusion.Candidate genes were verified by qRT-PCR.Furthermore,the methylated regulation pattern was confirmed in the BeWo cell demethylated model.Wnt7 a,expression regulated by DNA methylation,was initially screened out.4.Expression pattern and methylation level of DNMTs in PE placental trophoblast cellsDNMTs expression in cytotrophoblast cells was significantly increased in PE placenta compared with controls.The primary cytotrophoblastic spontaneously fusion in PE was decreased.These results showed that the abnormal DNA methylation level of trophoblast and the decrease of spontaneously fusion ability was shown in PE trophoblast cells.The abnormal expression of DNMTs may affects the process of trophoblastic fusion in PE placenta,which may be involved in the occurrence of PE.Based on the above results,we concluded that DNMTs were downregulated and DNA methylation was decreased during trophoblastic fusion.DNA methylation may be involved in the process of trophoblastic fusion by activating phosphorylation of the CREB signaling pathway.Wnt7 a is a target factor regulated by DNA methylation during trophoblastic fusion.The abnormal expression of DNMTs affects trophoblastic fusion in PE placenta,which may be involved in the occurrence of PE.