circRNA3669 promotes goat endometrial epithelial cells proliferation via miR-26a/RCN2 to activate PI3K/AKT-mTOR and MAPK pathways

The development of receptive endometrium(RE) from pre-receptive endometrium(PE) for successful embryo implantation is a complex dynamic process in which the morphology and physiological states of the endometrial epithelium undergo a series of significant changes, including cell proliferation and apoptosis. However, the molecular mechanisms are not yet fully understood. In this study, a higher circRNA3669 level was observed in PE than in RE of goats. Functional assays revealed that this overexpression promoted the proliferation of goat endometrial epithelial cells(GEECs) by activating the expression of genes related to the PI3K/AKT-mTOR and MAPK pathways,thereby inhibiting apoptosis in vitro. Furthermore, circRNA3669 functioned as a competing endogenous RNA(ceRNA) to upregulate Reticulocalbin-2(RCN2) expression at the post-transcriptional level by interacting with and downregulating miR-26a in GEECs. In addition, RCN2, which is highly expressed in the PE of goats, was found to be regulated by β-estradiol(E2) and progesterone(P4). Our results demonstrated that RCN2 also affected the key proteins PI3K, AKT, mTOR, JNK, and P38 in the PI3K/AKT-mTOR and MAPK pathways, thereby facilitating GEECs proliferation and suppressing their apoptosis in vitro. Collectively, we constructed a new circRNA3669-miR-26aRCN2 regulatory network in GEECs, which further provides strong evidence that circRNA could potentially play a crucial regulatory role in the development of RE in goats.

Adenomyosis-derived extracellular vesicles endow endometrial epithelial cells with an invasive phenotype through epithelial-mesenchymal transition

Extracellular vesicles from highly metastatic tumor cells have been shown to mediate epithelial-mesenchymal transition(EMT)-related events in recipient cells.In endometrial epithelial cells,EMT processes are known to be involved in the development of adenomyosis.We aimed to investigate whether adenomyosis-derived extracellular vesicles(AMEVs)are able to induce an EMT process in endometrial epithelial cells.In this study,AMEVs were isolated from patients with adenomyosis and characterized by transmission electron microscopy,Western blot,and nanoparticle tracking.Primary endometrial epithelial cells(EECs)were derived from normal endometrium tissues from patients with leiomyoma and co-cultured with AMEVs in vitro.AMEV uptake was examined by fluorescence confocal microscopy.The invasion of EECs was confirmed by Transwell assay.Immunohistochemistry,Western blot,and qRT-PCR were performed on EECs to illustrate the expression levels of cytokeratin 19,E-cadherin,vimentin,and zinc finger E-boxbinding homeobox 1(ZEB1).The results indicated that the cellular fluorescence intensity gradually increased after 48 h of co-culture,but decreased after 72 h.After co-culturing with AMEVs for 72 h,EECs expressed significantly lower levels of cytokeratin 19 and E-cadherin,and significantly higher levels of vimentin and ZEB1.Together these results demonstrated that AMEVs induce an EMT process and enhance the invasion of EECs.These changes may contribute to the pathogenesis and progression of adenomyosis.

Differentiation of Human Adipose-Derived Stem Cells into Endometrial Epithelial Cells

Objective:This study aimed to investigate the differentiation of human adipose-derived stem cells(hASCs)into endometrial epithelial cells(EECs)under certain induction conditions and to a further step provide a promising approach for ASCs in clinical practice to the treatment of severe intrauterine adhesion.Methods:Four groups of hASCs were separately cultured as follows:in Group 1,hASCs were cultured in a control medium(5%fetal bovine serum[FBS]+α-minimum Eagle’s medium[α-MEM]);in Group 2,hASCs were cultured in an induction medium(5%FBS+α-MEM+[1×10-7 mol/L 17β-estradiol]+10 ng/mL transforming growth factorβ1[TGF-β1]+10 ng/mL epidermal growth factor[EGF]+10 ng/mL platelet-derived growth factor BB[PDGF-BB]);in Group 3,hASCs and human endometrium cells(hEMCs)were cocultured in the control medium;and in Group 4,hASCs and hEMCs were cocultured in the induction medium.Results:When cocultured with hEMCs,the morphology of hASCs became similar with EECs,and the addition of factors such as EGF,TGFβ,PDGF-BB,and 17β-estradiol promoted differentiation.This study,for the first time,demonstrated estrogen receptor(ER)αand ERβexpression in hASCs and preliminarily explored changes in ERα,ERβ,β-catenin,and H19 mRNA expression during hASC differentiation.Furthermore,we concluded that H19 mRNA expression was negatively correlated with differentiation,which is seemingly related to the estrogen signaling pathway.Conclusions:hASCs revealed the potential for differentiating to EECs when cocultured with hEMCs.