Wnt/β-catenin Signaling Pathway Is Critical To Estrogen-induced Epithelial-mesenchymal Transition In The Pathogenesis Of Endometriosis

Objective(1) To investigate the occurrence of epithelial-mesenchymal transition (EMT) in endometriosis.(2) To investigate the role of estrogen in the occurrence of EMT and the function of Wnt/β-catenin signaling pathway in estrogen mediated EMT.(3) To investigate the molecular mechanism of Wnt/β-catenin signaling regulated EMT in endometriosis.Methods(1) All eutopic and ectopic samples were collected from women with endometriosis. The normal endometria as control groups were collected from women who simply because of fallopian tubal infertility. The expression of N-cadherin, E-cadherin, Vimentin, Snail and P-catenin in 20 normal,20 eutopic and 20 ectopic endometrial samples were assessed by immunohistochemistry.(2) Analyzed of E-cadherin, N-cadherin, Vimentin, Snail and β-catenin in normal control, endometriosis eutopic and ectopic endometrium by qRT-PCR and Western blotting.(3) Isolated and cultured of human endometrial epithelial cells (EECs). Analyzed biomarker of EMT in EECs treated with different dose (10-6,10-8mol/L) of 17β-estradiol by qRT-PCR and Western blotting. The cell morphology is observed by microscope. Analyzed migration and invasion ability of cells by Transwell Assay.(4) Analyzed dephosphorylated P-catenin in EECs treated with 10-6mol/L 17β-estradiol by Western blotting. The amount of nuclear P-catenin analyzed by immunofluorescence.(5) Ishikawa cells instead of EECs. Analyzed biomarker of EMT and β-catenin in Ishikawa treated with different dose (10-6, 10-8mol/L) of 17p-estradiol by qRT-PCR and Western blotting.(6) P-Catenin siRNA abrogated Wnt/β-catenin signaling, analyzing biomarker of EMT in Ishikawa treated with 10-6mol/L 17P-estradiol by Western blotting and examined cells migration, invasion ability by Transwell Assay.(7) To determine whether the SNAIL promoter is in fact regulated by the β-catenin/TCF3 complex, we analyzed the activity of SNAIL promoter in Ishikawa by Luciferease assay and Chromatin Immunoprecipitation (ChIP) Assay.(8) An animal model that would mimic ectopic implantation of the endometrium was established. To investigate the effect of β-catenin siRNA on implantation of ectopic lesions, biomarker of EMT and dephosphorylated P-catenin were analyzed by Western blotting. MMP9 and VEGF in pre-transplantation eutopic endometrium and implanted endometrial lesions were analyzed by immunohistochemistry.Results(1) The expression of E-cadherin in ectopic endometrium is lower than normal control and eutopic endometrium(.P<0.05), while there is no significant discrepancy in the expression of eutopic endometrium and normal control of E-cadherin(P>0.05). The expression of N-cadherin and Vimentin in ectopic endometrium is higher than normal control and eutopic endometrium(P<0.05), while there is no significant discrepancy in the expression of eutopic endometrium and normal control of N-cadherin and Vimentin (P>0.05). The expression of β-catenin and Snail in ectopic endometrium is higher than normal control and eutopic endometrium(P<0.05), while there is no significant discrepancy in the expression of eutopic endometrium and normal control of β-catenin and Snail(P>0.05). The upregulation of β-catenin significantly correlated with that of Snail in ectopic samples.(2) QRT-PCR and Western blotting was shown that E-cadherin expression is decreased in ectopic endometrium compared to eutopic endometrium and normal control (P<0.05). Conversely, N-cadherin and Vimentin overexpressed in ectopic endometrium compared to eutopic endometrium and normal control (P<0.05). P-Catenin and Snail overexpressed in ectopic endometrium compared to eutopic endometrium and normal control (P<0.05).(3) Primary cultured EECs responded to 17p-estradiol with classical EMT changes (fibroblastoid phenotype, increased expression of N-cadherin, Snail, P-catenin and decreased expression of E-cadherin) and increased migration and invasiveness.(4) 17β-estradiol upregulates the dephosphorylated P-catenin and promotes the nuclear translocation of P-catenin in EECs.(5) Ishikawa cells responded to 17β-estradiol with classical EMT changes (fibroblastoid phenotype, increased expression of N-cadherin, Snail, β-catenin and decreased expression of E-cadherin).(6) 17β-Estradiol induced overexpression of N-cadherin, Snail and lower expression of E-cadherin were abrogated when Ishikawa cells were treated with E2 together with P-catenin siRNA by Western blotting.(7) Dual luciferase and chromatin immunoprecipitation assay showed SNAIL promoter is activated by the β-catenin/TCF-3 complex in Ishikawa, and 17β-estradiol enhances the effect.(8) β-Catenin deletion abrogates the 17β-estradiol-induced increase the incidence of implanted endometrial lesions. After 10 days, the incidence of ectopic lesions in negative siRNA+E2, β-catenin siRNA+E2 and control are 75%,25% and 0, respectively. After 21 days, the incidence of ectopic lesions in negative siRNA+E2,β-catenin siRNA+E2 and control are 64%,0 and 0, respectively. Western blotting assay showed decreased E-cadherin and increased N-cadherin expression were demonstrated in the implanted tissues from scrambled siRNA-treated mice compared with their pre-transplantation counterparts and lesions from β-catenin siRNA-treated mice. P-Catenin and Snail expression were consistent with N-cadherin expression. Immunohistochemistry Assay showed increased MMP9 and VEGF expression was demonstrated in the implanted tissues from scrambled siRNA-treated mice compared with their pre-transplantation counterparts and lesions from P-catenin siRNA-treated mice.Conclusion(1) Epithelial-mesenchymal transition may be involved in adhesion, invasion and metastasis of endometriosis, which participated in the pathogenesis of endometriosis.(2) 17β-estradiol plays a critical role in EMT occurrence in endometriosis. The mechanism may be through activation of Wnt/β-catenin signaling pathway, inhibits the expression of E-cadherin to promote the generation of EMT.