Studies On The Mechanism Of Estrogen-meidated Estrogen Receptor Alpha,Beta And G-protein Coupled Estrogen Receptor In The Pathogenesis Of Endometriosis

Part One The expression of ERα,ERβ and GPER in eutopic and ectopic endometrium of endometriosisObjective Immunohistochemistry(IHC)analyses of ERα,ERβ and GPER in different types of endometriosis were performed,in order to clarify the relationship between different estrogen receptor subtypes and different types of endometriosis.Methods1.Collect normal menstrual cycle endometrium,eutopic endometrium of endometriosis,Ovarian endometriosis(OVE),Deep Infiltrating Endometriosis(DIE)and Abdominal wall endometriosis(AWE);2.Immunohistochemical analyses of ERα,ERβ and GPER in the collected endometrium were performed.Result1.The expression of ERs in normal menstrual cycle:GPER was mainly expressed in the cytoplasm and membrane of human endometrium epithelium cells(EECs)and the expression in human endometrium stromal cells(ESCs)was weak.The expression of GPER was the highest in endometrial proliferation period and decreased in the period of secretory and menstrual.ERα and ERβ were mainly expressed in the nucleus,and the expression of ERα and ERβ were observed in both EECs and ESCs.The expression of ERα and ERβ decreased in the secretory phase and menstrual phase,and the ESCs decreased most significantly;2.The expression of GPER in different types of endometriosis:In eutopic endometrium of endometriosis,GPER was expressed in the cytoplasm and membrane of both EECs andESCs and the expression was significantly higher than the normal endometrium.In the three types of endometriosis,the expression of GPER increased,the highest expression was seen in AWE.In OVE,GPER mainly expressed in the cytoplasm,while some entered into the nuclear.But in AWE and DIE,GPER obviously expressed in the nuclear,and nuclear expression is even higher than the cytoplasm and membrane expression;3.The expression of ERa in different types of endometriosis:The expression of ERa in eutopic endometrium was significantly higher than that in normal endometrium.In OVE and DIE ERa expression increased,while in AWE ERa expression decreased;4.The expression of ERβ in different types of endometriosis:The expression of ERβ in eutopic endometrium was significantly higher than that in normal endometrium.In all three types of endometriosis,ERβ expression increased,and in DIE of the most obvious.Conclusion1.Expression of ERα,ERβ and GPER is associated with normal menstrual cycle and is regulated by estrogen;2.ERα,ERβ and GPER were differently expressed in different types of ectopic endometrium,indicating that they play different roles in different types of endometriosis.Part Two Estrogen stabilizes hypoxia-inducible factor la through G protein-coupled estrogen receptor 1 in eutopic endometrium of endometriosisObjective1.Identify whether eutopic endometrium of endometriosis(EuEM)expresses higher level of HIF-1α protein than normal control endometrium(CoEM)and its relationship with GPER;2.To investigated whether GPER stabilized HIF-1α under estrogen stilulation in EuEM;3.To investigate whether HIF-1α signaling pathway can promote the invasion,metastasis,and angiogenesis of ESCs.Methods1.IHC detects the expression of GPER and HIF-1α in CoEM and EuEM;2.Isolation,culture and identification of ESCs;3.The expression of GPER and HIF-1α in ESCs was detected by Western blot and RT-PCR under 17-E2 stimulation;4.ESCs were stimulated by GPER antagonist-G15 for half an hour beforehand in order to inhibit the effect of GPER,cells were then treated with 17-E2 or G1.The expression of GPER and HIF-1α were detected by Western blot and RT-PCR.The expression activity of MMP9 and VEGF were detected by luciferase assay;5.ESCs were stimulated by GPER antagonist-G15 for half an hour beforehand in order to inhibit the effect of GPER,cells were then treated with 17-E2 or G1.The angiogenic ability of ESCs was detected by tube formation.The invasion ability of ESCs was detected by transwell assay.Results1.Compared with CoEM,the expression of GPER and HIF-1α protein in EuEM increased;2.We successfully separated ESCs,cell purity and positive rate were higher than 95%;3.Estrogen and G1 can simultaneously promote the transcription and translation of GPER in ESCs,and increase the level of GPER mRNA and protein.There is no effect ofEstrogen and G1 on HIF-1 a mRNA expression,but it can inhibit the degration of HIF-1α and active the HIF-1 a pathway,which promote the expression of HIF-1αdownstream target genes VEGF and MMP9.G15 can block these effects;4.Estrogen and G1 can promote ESCs angiogenesis,invasion,and metastasis,G15 can block these effects.ConclusionGPER stabilizes HIF-1α and thus promotes HIF-1α—induced VEGF and MMP9 in ESCs,which play critical roles in endometriosis.Part Three Intracellular Wnt/β-catenin Signaling Underlying estrogen-Induced MMP9 and VEGF Expression in EndometriosisObjective1.To clarify whether Wnt/β-catenin signaling is active in OVE;2.To study the role of Wnt/p-catenin signaling in estrogen-induced ESCs invasion and angiogenesis;3.To elucidate whether estrogen activates Wnt/β-catenin signaling through ERa and its molecular mechanism;4.To elucidate the molecular mechanism of Wnt/β-catenin signaling up-regulating MMP9 and VEGF to promote ectopic endometrial invasion and angiogenesis.Methods1.IHC,Western blot and RT-PCR were used to detect the expression of ERα,β-catenin,VEGF and MMP9 in CoEM,EuEM and ectopic endometrium(EcEM)of OVE;2.17-E2 stimulated ESCs,IF were used to observate the expression and localization of(3-catenin,Western blot,RT-PCR were used to detect the expression of VEGF and MMP9 expression;3.ERa overexpression vector,β-catenin overexpression and silencing vectors were constructed,transfected to T HESCs,and then detected β-catenin,VEGF and MMP9 expression,observated ESCs migration and invasion ability;4.Constructed ERa overexpression vector and β-catenin promoter luciferase reporter.Luciferase assay and Chromatin Immunoprecipitation(ChIP)were used to study the molecular mechanism of estrogen mediated Wnt/β-catenin signaling through ERa;5.Constructed VEGF and MMP9 promoter luciferase reporters,β-catenin overexpression and silencing vectors.Luciferase assay and ChIP were used to study the molecular mechanism of Wnt/(3-catenin signaling up-regulating the expression of VEGF and MMP9.Result1.Compared with CoEM,ERα,β-catenin,VEGF and MMP9 of EuEM and EcEM from OVE were higher expressed;2.Estrogen stimulated the expression of(3-catenin,induced(3-catenin into the nucleus,activated Wnt/β-catenin signaling,up-regulated the expression of MMP9 and VEGF,promoted the migration,invasion and angiogenesis ability of ESCs;3.ERa can bind to the ERE region of β-catenin promoter as a transcription factor to promote the expression of β-catenin in T HESCs;4.After binding to TCF3/LEF1,P-catenin binded to the TCF3 binding region of VEGF and MMP9 promoters,up-regulated the expression of VEGF and MMP9 in T HESCs.Conclusion Estrogen activates Wnt/β-catenin signaling through ERa,and then β-catenin binds to the nuclear transcription factor TCF3/LEF1 to initiate the downstream target gene VEGF,MMP9,which play important roles in the invasion,metastasis,and angiogenesis of endometriosis.Part Four Estradiol induces mesenchymal epithelial transition(MET)by regulating HIF-la pathway through ERβ to promote the formation of ectopic endometrium Objective1.To clarify the role of MET in the formation of ectopic lesions in endometriosis;2.To clarify the role of ERβ and HIF-1α pathway in the generation of e ectopic lesions in endometriosis;3.To elucidate the molecular mechanism of estrogen-mediated ERβ regulation of HIF-1αpathway-induced MET in endometriosis.Method1.IHC was used to detect the expression and localization of ERβ,HIF-1α,and MET marker E-cadherin,Vimentin,and E-cadherin transcription factor Twist in CoEM,EuEM and EcEM of OVE;2.17-E2 stimulated ESCs,The expression of ERβ,HIF-1α,E-cadherin,Vimentin and Twist were detected by Western blot and RT-PCR;3.17β-E2 stimulated ESCs for different time(0-7day)under hypoxia environment(1%O2 concentration),the expression of ERP,HIF-1α,E-cadherin,Vimentin,and Twist were detected by Western blot and RT-PCR;4.17β-E2 and(or)PHTTP stimulated ESCs.ICC was used to observe the changes of cellmorphology.Transwell assay was used to examin the migration ability of ESCs,and clone assay was used to detectthe proliferation ability of the cells;5.Constructed ERβ overexpression or silencing vector,transfected to T HESCs,and then detected ERp,HIF-1α,E-cadherin and Vimentin expression;6.Constructed HIF-1α promoter luciferase reporters.Luciferase assay and ChIP wereused to study the molecular mechanism of ERβ inhibition of the expression of HIF-1α.Result1.MET phenomenon is prevalent in EcEM of OVE;2.Estrogen can significantly promote the expression of ERβ and MET-related proteins in vitro and inhibit the expression of HIF-1α,which is still present even in hypoxicenvironment;3.Estrogen prolonged stimulation can significantly change the ability of ESCs invasion and clonal proliferation ability;4.ERβ can bind to the ERE region of HIF-1α promoter as a transcription factor to inhibit the expression of HIF-1α in T HESCs.ConclusionEstrogen inhibits HIF-1α pathway through ERβ to induce MET and promotes proliferation of EcEM and the formation of endometriotic lesions.