The Function Of M6A Methyltransferase METTL3 In Endometrial Stroma Decidualization

Endometrium decidualization is a key event in the entire reproductive process of embryo implantation,placenta complete development,fetal growth and final successful pregnancy.Endometrial decidual tissue not only provides suitable microenvironment for embryo implantation,but also protects the semiallogeneic fetus from rejection by maternal immune system.At the same time,it also can promote the development of the placenta,and provide enough nutrients to the fetus before the mature placenta formation.Once the decidualization of the endometrial tissue is obstructed,it may lead to adverse pregnancy outcomes,such as abnormal embryo implantation,impaired placental development and pregnancy loss.A variety of reproductive diseases,such as endometriosis,recurrent abortion,and antiphospholipid syndrome,are closely associated with decidual damage,although they differ pathologically.However,it remained unclear about the basic physiological mechanism of endometrium decidualization,which,for the greater part,place restrictions on our exploration of the pathological mechanism of pregnancy-related diseases caused by abnormal decidualization.Studies have confirmed that Methyltransferase-like 3(METTL3)mediated N6methyladenosine(m6A)modification in RNA is involved in the biological processes of carcinogenesis,spermatogenesis,angiogenesis,metabolism and early embryonic development by regulating cell proliferation,differentiation,apoptosis and invasion.Studies have shown that m6A modification have a vital role in endometrial cancer and adenomyosis.However,the basic regulatory role of m6A in the endometrial decidualization has not been studied so far,which prompted us to explore the physiological regulatory mechanism of m6A in endometrial stroma decidua.In our study,firstly we detect the expression and location of METTL3 in different periods of endometrial tissue in menstrual cycle.The results showed METTL3 is expressed in the proliferative and secretive endometrial tissue,and also present in decidual tissues.In the state of proliferation and differentiation,it shows METTL3 expression in primary and immortalized endometrial stromal cells(HESCs)cultured in vitro,which suggests METTL3 may participate in regulating the function of the endometrial stromal cells.Subsequently,siRNA and shRNA were used to knock down METTL3 in immortalized HESCs.By detecting Ki67 and EdU signal,it was found that METTL3 did not affect the proliferation of HESCs,but significant changes were found in expression level of decidualization marker gene IGFBP1 and PRL,suggesting that METTL3 have a critical role in regulating decidualization of HESCs.Further studies on the molecular mechanism will be carried out in the future,aiming to reveal the regulatory mechanism of m6A modification mediated by METTL3 in the decidualization,which can provide clues for the forecast and treatment of the complications caused by abnormal decidua in pregnancy.Estrogen(E2)regulates multiple biological functions mainly through its specific receptor,namely estrogen receptor(ER).ER can activate the transcriptional process and/or signaling events that control gene expression by binding to E2.The role of ER can be mediated by directly binding to the responsive element in gene promoter(genomic effect),or not directly binding to DNA(non genomic effect),or independent of ligand,which give rise to downstream signal changes.Whether through direct action,indirect action,or a combination of the two,the effect of estrogen on gene expression is controlled by complex mechanisms that are highly regulated.There are two subtypes of estrogen receptor,ERa and ERβ,which are encoded by genes ESR1 and ESR2,respectively.In addition to full-length ERα,there are many splicing variants of ERα.There are four splicing variants of mRN A transcribed by ESR1 gene in mice,among which variants 1,2 and 3 all encode a full-length protein of ERα(599 amino acids,599aa),while variant 4 encodes a truncated form of protein(499 amino acids,499aa).The variant ERα(499aa)have a variable exon in the 3’coding region,resulting in a different encoding protein from that of Erα(599aa).Since ERa have a vital role in the establishment of mouse uterine pregnancy,studies have shown that the presence of splice variants in human or mouse uterine plays an important synergistic or antagonistic role on ERα(599aa),or showing a significant role in physiological and pathological conditions independently of ERα(599aa).The presence of ERα(499aa)in the uterus of mice during early pregnancy has not been reported.This study in mice uterine cDNA of early pregnancy for template,PCR amplification,product sequencing and overexpression of ER alpha(499aa),all of these demonstrate the splicing variants exist in the early pregnancy uterus of mice.qRT-PCR detect its expression in early pregnancy uterus of mice.The data of Dual luciferase reportor assay showed that ERa(499aa)don’t display obvrious transcriptional activity,and there no effect for the transcriptional function of ER alpha(599aa).