Effect Of CHMP3,a Member Of ESCRT System,on The Epithelial-Mesenchymal Transition Of A549 Cell And Its Molecular Mechanism

Objective: Idiopathic pulmonary fibrosis(IPF)is a common chronic,progressive,and fatal lung disease characterized by abnormal proliferation and remodeling of fibroblasts.The etiology of IPF is still not clear.The current widely accepted view is that idiopathic pulmonary fibrosis is caused by abnormal activation of damaged alveolar epithelial cells,resulting in proliferation of fibroblasts,fibroblast recruitment,and epithelial-mesenchymal transition.Lead to the deposition of extracellular matrix,abnormal repair of damage and reconstruction of lung structure.There is no effective treatment for IPF now.Therefore,clarifying its occurrence and development process is of great significance for the prevention and treatment of IPF.Epithelial-mesenchymal transition(EMT)refers to the process by which epithelial cells lose their phenotype and gain the phenotype of mesenchymal cells.This process is accompanied by the disappearance of cell polarity,depolymerization of intercellular junctions,and rearrangement of the cytoskeleton.Under normal physiological conditions,EMT is involved in a series of important life processes such as embryogenesis,organ development,wound repair,and tissue remodeling.Under pathological conditions,EMT can also be abnormally activated to enhance the cell’s ability to proliferate and metastasize,which is particularly evident during the development and progression of cancer.Recent studies have shown that EMT plays an important role in the progression of IPF,and EMT of damaged alveolar epithelial cells may be one of the origins of mesenchymal cells.Endosomal sorting complex required for transport(ESCRT)is a multi-subunit complex that recognizes and sorts ubiquitinated proteins.Its members include: ESCRT-0,ESCRT-I,ESCRT-II,ESCRT-III,and Vps4-Vtal cofactor family,widely involved in cytokinesis,viral budding,autophagy,membrane damage repair,intracellular signal transduction and many other cellular processes.CHMP3 is a member of the ESCRT-III family and forms the ESCRT-III subunit with three other core subunits(Vps20,Snf7,and Vps2)and three regulatory subunits(Did2,Vps60,and Ist1)in a multivesicular body(MVB)work together in biosynthesis.Studies have shown that ESCRT system is involved in regulating receptor signaling pathways,maintaining epithelial cell polarity and inhibiting tumor cell metastasis.These phenomena are closely related to the cellular EMT process.We boldly speculated that the ESCRT system may play a role in the EMT process of alveolar epithelial cells.So we design experiments and explore.In this study,TGF-β was used to induce human lung adenocarcinoma cell line A549.The EMT model of alveolar epithelial cells was constructed.The expression of E-cadherin,Vimentin and α-SMA before and after the induction was detected,and the alveolar epithelial cells EMT were detected.The ESCRT system member CHMP3’s expression trend during the process.Using the MVB inhibitor U18666 A and the method of overexpression of CHMP3 A549 stably transfected cell lines,the effects of CHMP3 on the EMT progression of A549 cells and their potential molecular mechanisms were preliminarily explored.Methods: 1.In this study,We used TGF-β to induce A549 cells to construct alveolar epithelial cells EMT model.The changes of cell morphology before and after induction were observed by inverted microscope,and the changes of E-cadherin and Vimentin markers were detected by Western Blot before and after induction.E-cadherin,Vimentin and α were detected by immunofluorescence staining before and after induction.-Changes in expression of SMA to assess modeling efficiency.Western Blot technique was used to detect the changes of CHMP3 expression before and after induction,and the response of ESCRT system during cell EMT was observed.2.The inhibitory model of ESCRT system expression was constructed by interfering A549 cells with MVB inhibitor U18666 A.Western Blot was used to detect the changes in the expression of TSG101,CHMP1 B,CHMP3 and VPS4 in the ESCRT system under U18666 A intervention conditions,and evaluate the intervention efficiency.A549 cells were induced by TGF-β induction,U18666 A intervention and TGF-β/U18666 A combination,and the expression of Ecadherin and Vimentin before and after induction in each group was detected by Western Blot,and the inhibition of EMT process of alveolar epithelial cells by ESCRT system members was evaluated.Impact.3.We constructed A549 stably transfected cell line overexpressing CHMP3,induced A549 cells and A549 over-expressed CHMP3 cells using TGF-β,and detected the expression of E-cadherin and Vimentin before and after induction by Western Blot.The changes were evaluated by overexpression of CHMP3 on the EMT process in alveolar epithelial cells.Results: 1.Successfully constructed an alveolar epithelial cell EMT model.Compared with the control group,the morphological changes of the TGF-β induced group were significantly changed from the original cobblestone-like epithelial cell phenotype to the slender spindle-like interstitial cell phenotype.Western Blot results showed that the expression of E-cadherin was increased in the TGF-β induced group,and the expression of Vimentin was decreased.The results of immunofluorescence showed that the expression of E-cadherin and α-SMA increased in the TGF-β induced group,and the expression of Vimentin decreased.Western Blot results showed that the ESCRT system member CHMP3 expression was reduced in the TGF-β induced group.2.A549 stably transfected cell line overexpressing CHMP3 was successfully constructed.Western Blot detection revealed expression of exogenous CHMP3.The expression of E-cadherin was decreased in overexpression of CHMP3 group,and the increase of Vimentin expression was weaker than that of normal A549 cells.3.The expression of ESCRT system members in A549 cells was inhibited to a certain extent after intervention with inhibitor U18666 A.Western Blot results showed that the expression of CHMP3,CHMP1 B,and VPS4 were reduced but TSG101 expression was unchanged.The expression of E-cadherin was down-regulated in U18666A-induced group,and the expression of Vimentin was up-regulated.However,the change trend was not significant in the TGF-β-induced group,and there was no significant change in the TGF-β/U18666A-induced group compared to the TGF-β-induced group.Conclusion: The alveolar epithelial cell EMT model was successfully constructed by inducing A549 cells using TGF-β,and the ESCRT system member CHMP3 expression was reduced during this process.overexpression of CHMP3 promoted EMT in alveolar epithelial cells.Multivesicular bodies generation inhibitor U18666 A down-regulating CHMP3 expression did not promote EMT in alveolar epithelial cells.