Adenomyosis-derived Extracellular Vesicles Induced Epithelial-mesenchymal Transition Of Endometrial Epithelial Cells

BackgroundAdenomyosis is characterized as endometrial glands and stroma deep within the myometrium.However,the precise pathogenesis of adenomyosis is still unclear.More and more studies have shown that the epithelial-mesenchymal transition(EMT)process of endometrial epithelial cells(EECs)is involved in the pathogenesis of adenomyosis.EMT endow EECs invasion and migration ability.And EECs with invasion and migration ability is thought to be a prerequisite for the original establishment of adenomyosis lesions.EVs can induce EMT in recipient cells during cancer progression.Extracellular vesicles(EVs),are shed by prokaryotes and eukaryotic cells,and represent a new form in mediating cell-cell communication.EVs have been isolated from human uterine,human uterine epithelial cells and human endometriotic stromal cells.EVs play important roles in mediating cell-cell communication and participate in the pathogenesis of a range of diseases,including cancer.Cell-derived vesicles are thought to contribute to homeostasis,disease development and progression.EVs from the invasive potential donor cells can deliver content that causes the recipient cells to gain invasiveness.Notably,EVs isolated from diverse cell models have been identified to induce EMT in recipient cells.In this study,adenomyosis-derived extracellular vesicles(AMEVs)were isolated from adenomyosis tissue.In vitro,AMEVs induced endometrial epithelial cells(EECs)epithelial-mesenchymal transition(EMT).This process may be the results of transferring contents of AMEVs and the results of increasing HSPB1 and ZEB1 expression in EECs.Objective1.To define the structural characteristics and intrinsic protein cargoes of extracellular vesicles derived from adenomyosis2.To verified whether adenomyosis-derived extracellular vesicles can induce epithelial-mesenchymal transition in endometrial epithelial cells3.To analyze the mechanism of epithelial-mesenchymal transition in endometrial epithelial cells induced by extracellular vesicles derived from adenomyosis.Methods1.This study was approved by the Ethics Committee on human research of Chongqing Medical University and informed and signed written consent was obtained from all patients.Adenomyosis lesions and eutopic endometrium were collected during hysterectomy from 14 women with adenomyosis,while normal endometrium samples were collected during hysterectomy from 10 women with uterine myoma,who had not used oral contraceptives for the 3 months prior to tissue sampling.2.The freshly collected adenomyotic lesions were immediately ground and homogenized.AMEVs were isolated and purified from the homogenate by differential centrifugation and density gradient centrifugation.Transmission electron microscopy,low vacuum scanning electron microscopy and Nanoparticle tracking analysis were used to analyze the morphology and size distribution of AMEVs.Liquid chromatography-mass spectrometry and Western blotting were used to analyze the protein content and membrane markers of extracellular vesicles derived from adenomyosis tissue.3.Databases excavating,literature searches and protein function analysis were used to analyze the proteins in AMEVs associated with epithelial-mesenchymal transition and its downstream pathway.4.The endometrial epithelial cells were isolated from homogenate of endometrial tissue by double filtration through filters and by using different adhesion times of epithelial cells to stromal cells.CCK-8 was used to detect the cell viability of EECs after cocultured with AMEVs for 72 hours.Confocal microscopy was used to observe the uptake of AMEVs by EECs.Western blot analysis was used to confirm the changes of Cytokeratin 19,E-cadherin and Vimentin expression in EECs after treatment with AMEVs for 72 hours.Immunohistochemistry was used to identify the changes of CK19,E-cadherin,Vimentin,HSPB1 and ZEB1 in EECs after treatment with AMEVs for 72 hours.Flow cytometry was used to detect cytochalasin D-mediated inhibition of AMEVs uptake in EECs.Immunohistochemistry was used to identify the changes of HSPB1 after inhibition.Results1.Nanoparticle tracking analysis revealed that the AMEVs at 100,000 × g had a mean diameter of 221.0±8.3 nm and TEM showed that AMEVs had a lipid bilayer and a round shape.Western blot analysis showed that AMEVs expressed flotillin-2,CD 9 and CD 63,and mass spectrum showed that AMEVs contains 2,579 different proteins.2.We combined protein function analysis and literature searches with the uniport database to analyze and identify 20 EMT-related proteins in AMEVs.Of these proteins,HSPB1 had the highest emPAI(44.96)while Annexin A2 had a Ranked eighth emPAI(23.25).HSPA5 and MSN had an emPAI>5,S100A4 and EZR had an emPAI≥1,and 14 proteins had an emPAI<1: CTBP1,CTNNB1,PDCD6,PPP3R1,PEF1,CTBP2,TRIM28,RTN4,DDX17,CCAR2,GSK3 B,PDCD4,BMP7 and ENG.3.EECs are able to internalize AMEVs,and this uptake process can be partially blocked by cytochalasin D.AMEVs enhanced the viability(P < 0.001)and proliferation rate(P < 0.001)of EECs.when exposed to DiO-labeled AMEVs within 24 hours,fluorescence intensity of DiO-labeled AMEVs in EECs gradually increased.Fluorescence was mainly concentrated in the cytoplasm and predominantly in the subcellular compartments of EECs.When co-culture time extended to 72 h,the fluorescence intensity of DiO-labeled AMEVs inside and outside of the EECs gradually decreased.4.When cocultured with AMEVs for72 hours,Transwell assay showed AMEVs significantly increased the migration and invasion of EECs compared to those in control group(p<0.001).After treatment with AMEVs for 72 hours,the expression of cytokeratin 19 and E-cadherin in EECs were significantly downregulated,and vimentin expression was significantly upregulated,compared to those in the controls.These changes are consistent with the changes in adenomyotic lesions that a significant trend of the downregulation of cytokeratin 19 and E-cadherin and the upregulation of vimentin was also observed in the tissue samples(P < 0.05,respectively).5.When cocultured with AMEVs for 72 hours,the expression level of HSPB1(P =0.005)and ZEB1(P =0.003)were significantly higher than those in control groups.The change of HSPB1 were consistent with changes in adenomyotic lesions that a significant trend of the upregulation of HSPB1 was also observed in the tissue samples.Cytochalasin D significantly slowed the elevation of HSPB1 compared to the control and non-blocked groups(P < 0.05).The expression changes of E-cadherin were negatively correlated with HSPB1(R = 0.894,P = 0.001)or ZEB1(R = 0.762,P = 0.01)before and after co-culture of AMEVs.Conclusion1.Adenomyosis tissue can secrete extracellular vesicles with a mean diameter of 221.0±8.3 nm,which is a round-shaped lipid bilayer membrane vesicle,and which express CD9,CD63 and Flotillin-2 membrane biomarkers.There were 2579 proteins in AMEVs,including 20 EMT-related proteins.2.In vitro,AMEVs improved the cell viability and proliferative ability of EECs compared to those of the controls,and promoted the invasion of EECs,and induced the Epithelial-mesenchymal transition of EECs.3.AMEVs induced epithelial-mesenchymal transition of endometrial epithelial cells by transferring the contents.Combined with protein function analysis,the epithelial-mesenchymal transition of endometrial epithelial cells may be associated with increased HSPB1 and ZEB1 after co-cultured with AMEVs.