Long Non-coding RNA AFAP1-AS1 Promotes The Epithelial-mesenchymal Transition Of Endometriosis Is Associated With Transcription Factor ZEB1

Object:1.Screening and validation of differential expression of long non-coding RNAs(lncRNAs)in endometriosis(EM).2.Derivation of epithelial-mesenchymal transition(EMT)in endometriosis.3.To explore the mechanismof the relationship between lncRNA AFAP1-AS1,EMT in endometriosis and ZEB1.Method : 1.We measured the expression of AFAP1-AS1,HOTAIR,KLKP1,HNF1A-AS1,H19,UCA1,MALAT-1 mRNA in ectopic and eutopic endometrial tissues from patients of EM and normal endometrial from patients without EM(patients of tubal infertility)by real-time PCR.2.We measured the expressions of EMT-related protein including ZEB1,E-cadherin,N-cadherin,Vimentin,Keratin by real-time PCR and immunohistochemistry respectively.3.Wetransfected ectopic endometriosis cell and ishikawa cell to knockdown AFAP1-AS1 by using lentivirus and observe the efficiency of the interference by qRT-PCR.4.We observed the changes of EMT-related gene,protein and small GTPs family genes,protein by real-time PCR and western blot.5.We detected the changes of the cell morphology and the ability included proliferation,migration,invasion and inflammation of ectopic endometriosis cell and ishikawa cell by MTT,EdU,wound healing,transwell,ELISA assay.6.We used luciferase reporter gene assay to study the effect of AFAP1-AS1 on the regulation of pGL3-P886 activity in the promoter site of the EMT-related transcription factor ZEB1 promoter.7.In vivo experiment,the effect of AFAP1-AS1 on endometriosis was studied by subcutaneous tumorigenesis in nude mice.Result:1.AFAP1-AS1 was high-expression in ectopic endometriosis cells than eutopic endometriosis cells and normal endometriosis cells.2.EMT phenomenon exists in EM.The results showed that the expression of E-cadherin and Keratin expression were obviously higher in eutopicendometriotic tissues than ectopic endometriotic tissues.Meanwhile the expression of Vimentin and N-cadherin were significantly higher in ectopicendometriotic tissues than eutopic endometriotic tissues.The expression of ZEB1 was higher in ectopic and in eutopic lesions than in normal tissues.3.The efficiency of lentivirus-infected is strong,and the efficiency of knockout AFAP1-AS1 is about 70%.4.Knockdown of AFAP1-AS1 affected the expression of EMT marker including the upregulation of E-cadherin,keratinand the down-regulation of N-cadherin,vimentinand ZEB1 and some changes of small GTPs family.5.Knockdown of AFAP1-AS1 changed the cell morphology from spindle fiber sample to polygon epithelioid and inhibited the ablility of proliferation,migration,invasion,expression of inflammatory cytokines.6.AFAP1-AS1 knockdown could significantlyinhibit the expression of pGL3-P886 in the promoter site of EMT-related transcription factor ZEB1.7.After down-regulation of AFAP1-AS1 expression,subcutaneous tumor formation in nude mice was significantly reduced.Conclusion:The expression of AFAP1-AS1 in endometriosis is significantly different.The knockdown of AFAP1-AS1 can inhibit the activity of E2-induced transcription factor ZTB1 promoter pGL3-P886,suggesting that AFAP1-AS1 may induce endometriosis and the pathogenesis may be associated with EMT.