Effect Of MiRNA-138 On Epithelial-mesenchymal Transition Of Airway Epithelial Cells

BackgroundBronchial asthma is a chronic disease characterized by airway inflammation,airway hyperresponsiveness and airway remodeling.The important marker of airway remodeling are airway epithelial cells shedding,adhesion connection between cells broken,extracellular matrix deposition,muscle and fibroblast proliferation,causing thickening of the basement membrane and epithelial fibrosis.The change of epithelial-mesenchymal transition(EMT)in airway epithelial cells is believed to be involved in this process.At present,it is believed that airway epithelial injury and abnormal repair is an important pathogenesis of airway remodeling in asthma,and airway remodeling may be closely related to the EMT of airway epithelial cells.EMT is the differentiation of epithelial cells to mesenchymal cells.Many studies have found that TGF-β1 increased in bronchoalveolar lavage fluid and airway wall in asthma,so TGF-β1 and airway remodeling is closely related to TGF-β1 and is an important cytokine induced by EMT.MiRNA is a negative factor to regulate the expression of important genes,through its seed sequences target mRNA UTR 3 ’end untranslated region by specific binding and inhibit the translation of genes.Research shows that miRNA-138 upregulation can inhibit the EMT of non small cell lung cancer cells such as A549 cell line.MiRNA-138 can inhibit epithelial cells undergoing EMT in lung cancer,whether miRNA-138 produce aberrant expression in the airway epithelial cells undergoing EMT and upregulating miRNA-138 can also inhibit airway epithelial cells undergoing EMT,so much as inhibiting airway remodeling? Therefore,this experiment aim at observing whether TGF-β1 could induce EMT and mi RNA-138 can prevent TGF-β1 induced airway epithelial cell undergoing EMT of murine airway epithelial cells,thus serving as the basis for the study of asthma airway remodeling mechanism.Object1.To verify whether the TGF-β1 can induce EMT in mouse airway epithelial cell line TC-1;2.To verify whether the miRNA-138 become abnormal expression in EMT;3.To verify whether the overexpression of miRNA-138 can inhibit EMTinducd by TGF-β1;MethodsWestern Blot detection of TGF-β1 induced change in EMT related phenotype protein in TC-1 cell line;RT-qPCR to detect the expression of miRNA-138.To carry Cy3 fluorescent fragments of miRNA-138 mimic transfected TC-1 cells.The transfection efficiency was observed by fluorescence microscopy and detect the expression of mi RNA-138 by RT-qPCR.At the same time give TGF-β1 stimulating miRNA-138 transfected TC-1 cells,Western Blot detection of EMT related protein markers of epithelial cells and mesenchymal cell marker protein changes.Results1.The expression of epithelial phenotype protein E-cadherin was decreased,and the expression of vimentin,α-SMA and collagen-Ⅰin TC-1 cells was increased in cells induced by 20ng/ml final concentration of TGF-β1;2.The expression of miRNA-138 was weaker than in the normal group after 10ng/ml stimulation of TGF-β1 in TC-1 cells 24h3.Up regulation of miRNA-138 expression after TC-1 transfection of mimic mi RNA-138.Treated with TGF-β1 stimulates and transfection miRNA-138 group epithelial cell phenotype expression of E-cadherin and mesenchymal cell phenotype vimentin and α-SMA,collagen-I and not given stimuli of TGF-β1 expression in the normal control group had no significant difference.Conclusions1.TGF-β1 stimulated TC-1 cell line,epithelial cell phenotype protein decreased,mesenchymal cell phenotypic protein increased,suggesting that TGF-β1 can induce epithelial cell EMT.2.TGF-1 stimulates TC-1 cell lines after EMT that miRNA-138 downregulated;and mi RNA-138 overexpression can inhibit EMT of TC-1 cell strains induced by TGF-β1,suggesting that miRNA-138 in TGF-β1 induced airway epithelial cells EMT has an important role,the upregulation of miRNA-138 can inhibit EMT in airway epithelial cells and airway remodeling in asthma pathogenesis and prevention research to provide ideas.