Study On The Role And Mechanism Of Hypoxia-induced Autophagy In The Pathogenesis Of Endometriosis

Part ? The expression of HIF-la,LC3,E-cadherin and Vimentin in clinical specimens of endometriosisObjectiveWe aimed to exam the expression patterns of autophagy marker gene Microtubule-associated protein 1A/1B-light chain 3(LC3),and the relationship between LC3,Hypoxia-inducible factor-1?(HIF-1?)and epithelial-mesenchymal transition(EMT)marker E-cadherin and Vimentin in clinical specimens of endoemtriosis.MethodsCollect normal proliferative cycle endometrium(31 cases),eutopic(30 cases)and ectopic endometrium(30 cases)of endometriosis.The expression and location of LC3,HIF-1?,E-cadherin and Vimentin were detected by Immunohistochemistry(IHC).The protein expression level of LC3,HIF-1?,E-cadherin and Vimentin was monitored by Western blot assay.ResultsImmunohistochemical staining results:(1)LC3 protein was mainly localized in the cytoplasm of epithelial and stromal cells.LC3 expression level was significantly higher in ectopic endometrium with endometriosis.(2)The expression of HIF-1? was localized in the nucleus and cytoplasm in both epithelial and stromal cells.The expression level of HIF-1? was elevated in ectopic endometrium with endometriosis when compared with normal and eutopic endometrium.(3)E-cadherin was localized in the membrane of epithelial cells.The expression level of E-cadherin was dramatically decreased in ectopic endometrium with endometriosis when compared with normal and eutopic endometrium.(4)Vimentin expression was localized in the cytoplasm of epithelial and stromal cells.The expression of Vimentin was increased in ectopic endometrium with endometriosis.Western blot results show that compared with normal endometrium and eutopic endometrium from patients with endometriosis,the protein expression level of LC3,HIF-1? and Vimentin were elevated in ectopic endometrium with endometriosis,while the protein expression of E-cadherin was decreasedConclusionThe expression level of LC3 was significantly increased in the ectopic endometrium with endometriosis,and positively correlated with HIF-1? and Vimentin,negative correlated with E-cadherin.These results suggest that hypoxia may contributes to autophagy induction,which in turn participates EMT progress in endometriosisPart ? Hypoxia-inducible factor-1? promotes endometrial stromal cells migration and invasion by upregulating autophagy in endometriosisObjective1.Identify whether hypoxia promotes autophagy of endometrial stromal cells via HIF-1?signaling pathway2.To investigated the role of HIF-1? and autophagy in hypoxia induced migration and invasion of endometrial stromal cellMethods1.Isolation and cultivation of human primary endometrial stromal cells(ESCs)2.ESCs were treated by hypoxia for various time points,and then acidic vesicular organelles(AVOs)was detected by acridine orange(AO)staining and monodansylcadaverine(MDC)staining,autophagosome was detected by Transmission electron microscopy(TEM),the protein expression level of HIF-1?,LC3,Beclinl and P62 were measured by Western blot assay3.ESCs was transfected with HIF-1? overexpression plasmid and HIF-1? siRNA,then the protein expression of HIF-1?,LC3 and Beclinl were detected by Western blot assay,GFP-LC3 aggregates were observed with confocal microscopy4.ESCs was transfected with HIF-1? overexpression plasmid and HIF-1? siRNA under normoxia and hypoxia condition,then Transwell migration and invasion assay was performed5.ESCs was transfected with Scramble siRNA and Beclinl siRNA under normoxia and hypoxia condition,then Transwell migration and invasion assay was performed6.ESCs was treated with chloroquine diphosphate salt and 3-Methyladenine under normoxia and hypoxia condition,then Transwell migration and invasion assay was performedResults1.Human primary endomertrial stromal cells were isolated successfully and the purity was above 95%2.Hypoxia could promote autophagy through regulation of HIF-1? signaling in endometrial stromal cells3.Inhibition of HIF-1? and autophagy could attenuate hypoxia triggered migration and invasion of endometrial stromal cellConclusionHypoxia promotes endometrial stromal cells migration and invasion through HIF-1?signaling denpendent autophagyPart ? Long non-coding RNA MALAT1 mediates hypoxia-induced pro-survival autophagy of endometrial stromal cells in endometriosisObjective1.To clarify the role of LncRNA MALAT1 in hypoxia induced HIF-1? dependent autophagy in endometrial stromal cells.2.To elucidate the effect of LncRNA MALAT1 dependent autophagy on endometrial stromal cells apoptosis under hypoxia condition.Methods1.Isolation and cultivation of human primary endometrial stromal cells(ESCs)(See Part?).2.The expression of LncRNA MALAT1 in endometriosis tissues was detected by RT-PCR.3.Overexpression and silencing of HIF-1?,respectively.Then the protein expression of HIF-1? was detected by Western blot,the expression of LncRNA MAALT1 was measured by RT-PCR.4.ECSs were transfected with scramble siRNA and MALAT1 siRNA,then the expression levels of HIF-1? and LC3 were evaluated by Western blot,GFP-LC3 dots were observed under confocal microscopy,autophagosome was detected by Transmission electron microscopy(TEM).5.ECSs were transfected with Beclinl siRNA or treated with 3-MA,then the protein expression level of caspase-3,Bax and Bcl-2 were detected by Westrn blot,cell apoptosis rate was detected with flow cytometry,apoptotic changes were detected by Hoechst33342 staining.Results1.The expression level of LncRNAMALATI,HIF-1? and LC3 was significantly elevated in ectopic endometrium with endometriosis,when compared with normal endometrium and eutopic endometrium with endometrisois.2.Hypoxia upregulates the expression of LncRNA MALAT1 in a HIF-1? dependent manner.3.Hypoxia promotes autophagy through upregulation LncRNA MALAT1.4.Inhibition of hypoxia induced autophagy promotes endometrial stromal cells apoptosis.ConclusionHypoxia promote autophagy through HIF-1? dependent LncRNA MAALT1 upregulation and hypoxia induced autophagy act as a pro-survival mechanism in endometrial stromal cells.Part IV Autophagy contributes to hypoxia-induced epithelial to mesenchymal transition of endometrial epithelial cells in endometriosis Objective1.To clarify whether hypoxia induces epithelial-mesenchymal transition(EMT)in endometrial epithelial cells(EECs)2.To evaluated whether hypoxia promotes autophagy via HIF-1? signaling pathway in endometrial epithelial cells3.To determine the role of autophagy in hypoxia triggered endometrial epithelial cells EMT,migration and invasionMethods1.EECs were cultured under hypoxia for various time points,then protein expression of E-cadherin,Vimentin and N-cadherin were detected by Western blot,cell morphology was observed under inverted microscope,immunocytochemistry was performed to detect the expression of E-cadherin and Vimentin2.EECs were cultured under hypoxia for various time points,then protein expression of HIF-1?,LC3 and Beclinl were detected with Western blot,autophagosome formation was observed by transmission electron microscopy(TEM).Ishikawa cells were treated by normoxia or hypoxia,then protein expression of HIF-1?,LC3 and Beclinl were detected with Western blot,GFP-LC3 dots were observed under conforcal microscopy Then Ishikawa cells were transfected with HIF-1? overexpression plasmid and siRNA,then the protein expression of HIF-1?,LC3 and Beclinl were detected with Western blot again3.EECs or Ishikawa cels were treated with 3-MA,CQ or Beclinl siRNA respectively,then protein expression of Beclinl,LC3,E-cadherin and Vimentin were detected with Western blot,cell migration and invasion abilities were detected by Transwell assayResults1.Hypoxia could trigger EMT and promotes cell migration and invasion in endometrial epithelial cells2.Hypoxia promotes autophagy via HIF-1? signaling pathway in endometrial epithelial cells3.Inhibition of autophagy could decrease hypoxia induced EMT and cell migration and invasion in endometrial epithelial cellsConclusionHypoxia promotes autophagy through HIF-1? pathway to induce EMT and enhance cell migration and invasion in endometrial epithelial cells.