MiR-200c Inhibit TGF-β1 Induced Epithelial To Mesenchymal Transition In Cell Model

BackgroundTracheal transplantation is an effective method for different types of diseases,such as primary airway stenosis,tracheal mucosa damage,and layngotracheal tumors.However,formation of a large number of fibroblasts and myofibroblasts after tracheal transplantation can block the lumen of tracheal.Therefore,the key to successful tracheal transplantation is the treatment of tracheal stenosis after transplantation.The major causes of stenosis of tracheal transplantation contain: excessive fibrosis and cartilage disorder.Using of sustanined-release bone morphogenetic protein and epidermal growth factor to promote the formation of tracheal cartilage,maintain the normal structure of trachea.But,there are a lot of unsolved problems,such as fibroblast and myofibroblast formation,airway implants experience immune rejection,inflammatory cell invasion,which tremendously interfere with tracheal transplantation reconstruction.The source of these fibroblasts and myofibroblasts are still unclear,but two major possible sources are bone marrow and transitioned from tracheal epithelial cells.Owing to tissue fibrosis is the main pathological changes in the process of tracheal transplantation.So these article will focus the source of these fibroblasts and myofibroblasts in transitioned tracheal epithelial.We identify these process epithelial to mesenchymal transition(EMT).Studies demonstrated that the EMT plays an important role in the pathogenesis of airway replacement.EMT results in a loss of epithelial properties and a gain of mesenchymal properties.Another factor involved in formation of fibroblasts and myofibroblasts of organ is transforming growth factor-β1(TGF-β1).TGF-β1 conbine receptor to catalyze protein phosphorylation,regulate expression of inhibitors of collagen and metalloproteinases transcriptional genes,also inhibit extracellular matrix degradation,promote activation of fibroblasts.Therefore,TGF-β1 enhance EMT,leading to hyperplasis of fibrous tissue,and play an important role in the pathophysiology of stenosis of tracheal transplantation.Many previous studies have demonstrated that microRNAs,short 20-22 nucleotide noncoding RNAs,are very important in fibrous tissue proliferation,migration and other physiological processes,for example,myocardial fibrosis,pulmonary fibrosis,glomerular fibrosis,and tumor cell migration.Simultaneously miRNAs play an important role in EMT regulation.MiR-200 c,a member of the MiR-200 family,regulates the expression of EMT-related transcription factors through TGF-β pathway during the pathogenesis of other organ fibrosis.While there is little research on the regulation of MiR-200 c in the process of EMT in human bronchial epithelial cells(HBE).Therefore,we used a model of EMT in TGF-β1-stimulated HBE to determine whether MiR-200 c regulation has an effect on EMT.ObjectiveBuilding a cell model of EMT of HBE in vitro,simulate pathological changes of stenosis caused by hyperproliferation of fibrosis after tracheal transplantation.On the basis of the cell model,observe connection between the expression of MiR-200 c and EMT model of TGF-β1 induced epithelial cells.Investigating effect of MiR-200 c on treatment of stenosis.Laying theoretical foundation to prevent and treat stenosis after tracheal transplantation.Methods1.Same concentration of TGF-β1 induce epithelial cells in different times,RT-qPCR detects gene expression of EMT-associated markers;Western-blot and immnuofluorescence detect expression of EMT-associated proteins;Building a cell model of EMT of HBE in vitro,simulate pathological changes of stenosis caused by hyperproliferation of fibrosis after tracheal transplantation2.Upregulation or downregulation of MiR-200 c in epithelial cells,and then stimulated by TGF-β1,CCK-8 detects proliferation changes of cells,inspects morphology changes of cells,RT-qPCR detects gene expression of EMT-associated markers;Western-blot and immnuofluorescence detect expression of EMT-associated proteins.Results1.Same concentration of TGF-β1 induce epithelial cells in different times,such as 0h,24 h,36h,48 h,RT-qPCR suggests that EMT-associated gene markers change: expression gene of E-cadherin downregulates,while expression gene of Vimentin and α-SMA upregulate.Western-blot and immunofluorescence suggest that EMT-associated proteins change: expression of E-cadherin protein decreases,while expression of Vimentin and α-SMA increase.These results indicate that we build a cell model of EMT of HBE in vitro successfully,simulate pathological changes of stenosis caused by hyperproliferation of fibrosis after tracheal transplantation.2.Downregulation expression of MiR-200 c combine with and stimulation of TGF-β1,CCK-8 detects proliferation changes of cells,show that group of downregulation+TGF-β1 have stonger proliferation than group of stimulated by TGF-β1 alone;Inspects cells morphology under microscope,show that group of downregulation+TGF-β1 morphology tend to mesenchymal cells.Upregulation expression of MiR-200 c combine with and stimulation of TGF-β1,CCK-8 detects proliferation changes of cells,show that group of upregulation+TGF-β1 have weaker proliferation than group of stimulated by TGF-β1 alone;Inspects cells morphology under microscope,show that group of upregulation+TGF-β1 morphology tend to cobblestone.3.Downregulation expression of MiR-200 c combine with stimulation of TGF-β1,RT-qPCR suggests that EMT-associated gene markers change,expression of E-cadherin in group of downregulation+TGF-β1 decreases more than the group of TGF-β1 stimulation alone,while expression of Vimentin and α-SMA in group of downregulation+TGF-β1 increase more than the group of TGF-β1 stimulation alone.4.Downregulation expression of MiR-200 c combine with stimulation of TGF-β1,Western-blot and immunofluorescence suggest that expression of E-cadherin protein change,expression of E-cadherin in group of downregulation+TGF-β1 decreases more than the group of TGF-β1 stimulation alone,while expression of Vimentin and α-SMA in group of downregulation+TGF-β1 increase more than the group of TGF-β1 stimulation alone.Upwnregulation expression of MiR-200 c combine with stimulation of TGF-β1,Western-blot and immunofluorescence suggest that expression of E-cadherin protein change,expression of E-cadherin in group of upregulation+TGF-β1 increases more than the group of TGF-β1 stimulation alone,while expression of Vimentin and α-SMA in group of upwnregulation+TGF-β1 decrease more than the group of TGF-β1 stimulation alone.ConclusionResults indicate downregulation of MiR-200 c enhanced TGF-β1 induced EMT in HBE,while upregulation of MiR-200 c can reverse this process.Our results identify MiR-200 c play an important role in pathophysiological process of fibroblasts and myofibroblasts.Speculate that MiR-200 c may play an important role in process of stenosis after tracheal transplantation,and it may have potential application in prevention and treatment stenosis after tracheal transplantation.