Studies On The Mechanism Of Estrogen-mediated Notch Signaling Involvement In The Development Of Endometriosis

Part I: Expression changes of Notch signaling pathway related molecules in eutopic and ectopic endometriumObjectiveTo determine the the relationship between Notch signaling and endometriosis by comparing the expression of NOTCH1,JAG1 and N1 ICD in eutopic and ectopic endometrium of patients with endometriosis and normal endometrium.Method1.Eutopic and ectopic endometrium of patients with endometriosis and normal endometrium of patients with tubal infertility were collected.2.Immunohistochemical staining(IHC)was used to deterimine the celllar distribution of NOTCH1,JAG1 and N1 ICD in the collected endometria while Western blot was used to quantitatively analyze the expression of these molecules.Result1.Distribution of Notch signaling related molecules in endometria: 1.NOTCH 1 and JAG1 were both expressed on the membrane of endometrial epithelial cells,and almost negatively expressed in stromal cells.(2)N1ICD was expressed in both epithelial cells and stromal cells.In normal and eutopic endometria,N1 ICD was predominently located in the cytoplasm of epithelial cells,while in ectopic endometrium,it was mainly expressed in the nuclei of epithelial cells and stromal cells.2.The expression of Notch signaling related molecules in endometria: Compared with normal endometrium and eutopic endometrium,the expression of NOTCH1,JAG1 and N1 ICD in ectopic endometria was significantly increased while there was no significant expresssion difference of these molecules between normal and eutopic endometria.Conclusion1.The increased expression of NOTCH 1,JAG1 and N1 ICD in ectopic endometrium of patients with endometriosis indicates that Notch signaling pathway is associated with the pathogenesis of endometriosis.2.The different expression and localization of NOTCH1,JAG1 and N1 ICD in ectopic endometrial epithelial cells and stromal cells suggest that Notch signaling plays different roles in endometrial epithelial and stromal cells,respectively.Part II: Estrogen promotes epithelial-mesenchymal transition of endometriotic epithelial cells through Notch signaling pathwayObjective1.To analysize the expression of EMT-related moleculars in eutopic and ectopic endometrium obtained from patients with endometriosis and to test whether EMT indeed occurred in endometriosis.2.To deterimine whether estrogen can promote the expression of Notch signaling related molecules and the occurrence of EMT in endometrial epithelial cells.3.To investigate the role of estrogen receptor(ER)4.in the expression of estrogen-induced Notch signaling pathway-related molecules and estrogen-induced EMT in endometrial epithelial cells.5.To investigate the role of Notch signaling in estrogen-induced EMT in endometrial epithelial cells.Method1.Normal human endometrial epithelial cells(EECs)were isolated,cultured and identificated.2.Serial sections derived from the samples in Part I were utilized to stain EMT-related molecules(E-cadherin and Vimentin)and determine their expression and celllar distribution in normal,eutopic and ectopic endometria.3.Afer incubation with 17 beta-estrodial,the expression of Notch signaling-related molecules(NOTCH1,JAG1 and N1ICD)and EMT-related molecules(E-cadherin,Vimentin and slug)were analysed by Western blot.The morphological changes of EECs were observed by phase contrast microscopy,and the capacity of migration and invasion of EECs were detected by Transwell assay.4.17 beta-estrodial was adminstrated following the blockage of estrogen receptors of EECs by estrogen receptor inhibitor ICI.Then,Western blot was used to analyse the expression of Notch signaling-related molecules and EMT-related molecules.Phase contrast microscopy was used to observe the morphological changes of EECs.Transwell assay was performed to detect the migration and invasion of EECs.5.17 beta-estrodial was adminstrated following the blockage of Notch signlaing by Notch signaling inhibitor DAPT.Then,Western blot was used to analyse the expression of Notch signaling-related molecules and EMT-related molecules.Phase contrast microscopy was used to observe the morphological changes of EECs.Transwell assay was performed to detect the migration and invasion of EECs.Result1.EECs were sucessfully isolated,and the purity and positive rate were higher than 95%。2.The expression of E-cadherin was downregulated while Vimentin was upregulated in ectopic endometria compared with normal endometria.Their expression in eutopic endometria are not significantly different from the normal endometria.3.Estrogen promoted the expression of NOTCH1,JAG1,N1 ICD,Vimentin and Slug in EECs,and surpressed the expression of E-cadherin in a time-dependent manner.Simultaneously,under the stimulation of estrogen,the shape of EECs was changed from ellipse to long spindle,and the capacity of migration and invasion was increased significantly.4.The expression of estrogen-induced Notch signaling-related molecules and Notch activity were decreased significantly after estrogen receptor blockage by ICI.As well,EMT was also reversed;5.Estrogen-induced EMT in EECs was reversed after inhibition of Notch signaling pathway by DAPT.Conclusion1.EMT occurred in EECs derived from ectopic endometria from patients with endometriosis.2.Through estrogen receptors,estrogen activates Notch signaling to promote EMT in EECs so as to involve in the invasion and metastasis of endometriosis.3.Notch signaling blockage may become a novel strategy to prevent the occurrence and development of endometriosis.Part III: Role and mechanism of estrogen-mediated Notch signaling in regualtion of invasion and metastasis of endometrial stromal cells in endometriosisObjective1.To determine whether Notch signaling pathway is activated or not in endometriosis.2.To determine the role of Notch signaling in estrogen-induced invasion of ESCs.3.To determine whether estrogen activates Notch signaling pathway through ER? in endometriosis and the corresponding mechanism.Method1.ESCs were sucessfully isolated and the purity and positive rate of ESCs were higher than 95%.2.After incubation with 17 beta-estrodial,the expression of NOTCH signal-related molecules(NOTCH1,JAG1 and N1ICD),MMP9 and VEGF in ESCs were analysed by Western blot.The nuclear translocation of N1 ICD was detected by immunofluorescence and immunocytochemistry.The migration and invasive ability of ESCs were detected by Transwell assay.3.17 beta-estrodial was adminstrated following the blockage of estrogen receptors of ESCs by estrogen receptor inhibitor ICI.Then the expression of NOTCH signal-related molecules,MMP9 and VEGF was analysed by Western blot.The migration and invasion of ESCs were analysed by Transwell assay.4.The human endometrial stromal cell line(HESCs)were transfected with constructed NOTCH1 sh RNA to block the Notch signaling in ESCs,then 17 beta-estrodial was adminstrated.Western blot was used to analyse the expression of NOTCH signal-related molecules and expression of MMP9 and VEGF.Transwell assay was performed to detect the migration and invasion of ESCs.Result1.Estrogen activates Notch signaling and promotes migration and invasion of ESCs in a time-dependent manner.2.After estrogen receptor inhibition by ICI in ESCs,estrogen-induced Notch signalingrelated molecules and expression of MMP9 and VEGF were blocked.3.After Notch signaling sclience in ESCs,estrogen-induced expression of MMP9 and VEGF was blocked.4.Estrogen regulates the transcription of Notch signaling key molecules through estrogen receptor elementsConclusionEstrogen activates Notch signaling pathway by receptors to upregulate the expression of VEGF and MMP9,and ultimately promotes migration and invasion of ectopic endometria.