Linc-ROR Promotes Epithelial-mesenchymal Transformation By Targeting MiR-204-5p/SMAD4 In Endometriosis

Background: Endometriosis(EMs)is a benign disease caused by the extra-uterine implantation of endometrium.The proposed transplantation theory and coelomic metaplasia theory partially reveal the pathogenesis underlying endometriosis.However,the detailed pathophysiology has not been elaborated and further researches are needed.In recent years,studies on epithelial-mesenchymal transition(EMT)suggested that the cellular transformation from epithelial morphology to mesenchymal morphology plays an important role in endometriosis.The microenvironment in ectopic lesions,including high level of estrogen,hypoxia and inflammation,has been proved account for the abnormal regulation of EMT in EMs.More importantly,the aberrant expressed non-coding RNAs were identified in EMs and it was proved that they regulate the EMT process via the manner of competitive endogenous RNA(ce RNA).Purposes: First part: to identify the aberrant expression of Linc-ROR in endometriosis,and explore the regulatory effect of Linc-ROR on ISK and E6E7 cells in vitro.Second part: to explore the possible downstream micro RNA of Linc-ROR,and verify the functional role of the micro RNA in vitro.Third part: to verify the combination of miR-204-5p and SMAD4,and the regulatory effect of Linc-ROR and miR-204-5p on SMAD4,and to confirm that Linc-ROR exerts cell function regulation through miR-204-5p/SMAD4 through rescue experiment.Forth part: construct an endometriosis nude mouse model to verify that Linc-ROR participates in endometriosis by regulating miR-204-5p/SMAD4 in vivo.Methods:First part: ectopic lesions and eutopic endometrium from patients with endometriosis,and normal endometrium from patients without endometriosis was collected,then q-RT-PCR analysis were performed to determine the differential expression of Linc-ROR between tissues.In ISK and E6E7 cell line transfected with Linc-ROR or vector,the CCK-8and Ed U assays were used to determine cellular proliferation,and transwell invasion assay was used to determine cellular invasion ability.What’s more,the western-blotting analysis on E-cadherin and Vimentin was conducted to confirmed the regulation of Linc-ROR on EMT process.Second part: expression level of candidate micro RNAs were first identified in tissues,and regulation of Linc-ROR was further confirmed in vitro.Dual-luciferase reporter assay was used to determine whether Linc-ROR functions as ce RNA of miR-204-5p.Then loss-and-gain-of function manner was conducted to prove the regulation of miR-204-5p upon cellular function and EMT process.Third part: combination between miR-204-5p and SMAD4 m RNA was confirmed using dual-luciferase reporter assay.Further,the expression level of SMAD4 m RNA together with SMAD4 protein expression were detected in different endometrium tissues.The regulation of Linc-ROR and miR-204-5p on SMAD4 was conducted in ISK and E6E7 cells.A rescue examination was conducted to prove that miR-204-5p was able to reverse the cellular changes brought by Linc-ROR in vitro.Forth Part: endometriosis was established in nude mice and intervened with si-Linc-ROR or si-NC.Expression level of miR-204-5p,SMAD4,Ki-67,E-cadherin and Vimentin were detected to verify the regulatory effect of Linc-ROR on miR-204-5p/SMAD4 in endometriosis.Results:First part: upregulated expression of Linc-ROR was observed in EC group compared to the NC and EU group.The results of the CCK-8 and Ed U assay verified the induced proliferation after Linc-ROR overexpression.The results of the transwell invasion assay revealed the increased invasion capability of ISK and E6E7 cells containing plasmid Linc-ROR compared to the control vector group.Upregulated vimentin and decreased E-cadherin were detected,which reflected the promotion of EMT by Linc-ROR.Second part: miR-204-5p showed significant downregulated changes in EC group comparing with the NC group.A significant decrease in miR-204-5p expression was detected after Linc-ROR overexpression.Dual-luciferase reporter assay confirmed Linc-ROR functions as ce RNA of miR-204-5p.Over expression of miR-204-5p inhibited cell proliferation and invasion as measured by the CCK-8,Ed U,and transwell invasion assays,while downregulation of miR-204-5p enhanced cell proliferation and invasion in ISK and E6E7 cells.Elevated expression of E-cadherin(E-CAD)and decreased expression of Vimentin(VIM)was confirmed in the miR-204-5p mimic group.Meanwhile,decreased levels of E-cadherin and increased levels of vimentin in the miR-204-5p inhibitor group presented a promotion of EMT.Third part: dual-luciferase reporter assay indicate that plausible regulation of miR-204-5p on SMAD4 m RNA.Elevated expression of SMAD4 in both RNA and protein level merged in EC group compared with NC group.Elevated SMAD4 expression was determined with Linc-ROR overexpressed.Meanwhile,upregulated miR-204-5p repressed the expression of SMAD4,decreased miR-204-5p promoted expression of SMAD4.Furthermore,the expression of SMAD4 was significantly down-regulated after cells were co-transfected with plasmid Linc-ROR and the miR-204-5p mimic.The enhanced proliferation and invasion promoted by Linc-ROR was reduced after cells were co-transfected with the miR-204-5p mimic.Concurrently,ISK and E6E7 cells transfected with Linc-ROR and the miR-204-5p mimic showed higher levels of E-cadherin and lower levels of vimentin compared with cells co-transfected with plasmid Linc-ROR and the mimic NC.Forth part: down-regulated SMAD4 expression and elevated miR-204-5p expression were detected in endometriosis mice model intervend with si-Linc-ROR.Concurrently,mice intervened with si-Linc-ROR showed higher levels of E-cadherin and lower levels of vimentin and Ki-67 compared with mice treated with si-NC.Conclusion: Linc-ROR acts as a ce RNA and promotes EMT process,cellular proliferation and invasion by targeting the miR-204-5p/SMAD4 axis in endometriosis.