Lyn Kinase Regulates CSE-induced Epithelial-mesenchymal Transition Through The Smad2/3 Pathway

Background Chronic obstructive pulmonary disease is a chronic inflammatory disease that is characterized by persistent respiratory symptoms and airflow limitation.Smoking is the most important environmental risk factor for chronic obstructive pulmonary disease and promotes changes in airway structure during disease progression.Epithelial-mesenchymal transition is one of the important pathogenesis of airway remodeling,which shows that the expression of epithelial markers is decrease and the expression of mesenchymal markers is increase.Multiple studies have shown that cigarette smoke extract can promote Epithelial-mesenchymal transition formation,but its specific mechanism is not elucidated.TGF-βand Smad2/3 signaling pathway are both carcinogenic and tumor-suppressing in tumor formation.Inflammatory microenvironment in neoplasms of TGF-β may cause cancer cell apoptosis and tumor inhibition,may also induce EMT to promote cancer cells invasion and metastasis.However,the fine regulatory mechanism of Smad2/3 signaling pathway in the process of EMT caused by smoking is still to be further studied.Non-receptor tyrosine kinase Lyn has a two-way regulatory role,and its subcellular localization may be the key to its function.Lyn kinase studies less in the mechanism of chronic obstructive pulmonary disease.Only a few studies have shown that knock-down Lyn can decrease cytotoxicity of CSE in airway epithelial cell.To explore the role of Lyn in CSE-induced EMT is of great significance to find a new drug therapy target for chronic obstructive pulmonary disease.In this study,we used the COPD mouse model and CSE-stimulated 16 HBE cells as the main research object.To investigate the role of Lyn kinase,Smad2/3 signaling pathway in CSE-induced EMT.Chapter One EMT induction by cigarette smoke extract and its signal transduction in miceObjective To investigate the induction of EMT mediated by cigarette smoke extract,and signal transduction in mice.Methods 10 C57BL/6J male mice were randomly divided into two groups: control group and COPD group.COPD mice model was establised by intranasal instillation of CSE and lipopolysaccharide.After modeling,total cell numbers and cell differentials in the BALF were determined.H&E staining and PAS staining of the mice lung tissue,to observe alveolar destruction and airway mucus secretion.The m RNA of TGF-β1 and MUC5 AC were detected by RT-q PCR.The concentration of TGF-β 1 was detected by ELISA.The expression of E-cadherin,Vimentin,α-SMA in the lung tissues were examined by immunofluorescence.The protein expression of E-cadherin,Vimentin,α-SMA,TGF-β1,Smad2/3 and p-Smad2/3,Lyn and p-Lyn in the lung tissues were detected by Westem blot.Results Compared with the control group,the COPD group significant increased number of total cells,neutrophils and macrophage in the bronchoalveolar lavage fluid.H&E staining showed that the mean linear intercept(MLI)and alveolar destruction index(DI)were significantly increased in COPD group.The number of goblet cells present in the airway epithelium of COPD mice exposed to CSE decreased compared with the control group.The levels of TGF-β1 and MUC5 AC m RNA were higher in the COPD group than in the control group.The concentration of TGF-β1 was significantly increased in COPD group.The expression of E-cadherin was decreased,and Vimentin,α-SMA,TGF-β1,Smad2/3 and p-Smad2/3,Lyn and p-Lyn were increased in the COPD group.Conclusion MUC5 AC was the main component of airway mucus hypersecretion in COPD mice.The TGF-β1 mediated Smad2/3 pathway may participate in airway EMT caused by CSE.Lyn kinase may be involved in Smad2/3 pathway regulate airway EMT caused by CSE.Chapter Two Regulatory mechanism of Lyn kinase in CSE-induced Epithelial-mesenchymal transition in 16 HBE cellsObjective To elucidate the mechanism of Lyn kinase in CSE-induced EMT in 16 HBE cells.Methods In part one,cultured 16 HBE cells were exposed to CSE at various concentrations and for defferent durations.The protein expression of E-cadherin,Vimentin and α-SMA were detected by Westem blot.In part two,Lyn-si RNA-treated 16 HBE cells were divided into four treatment groups: control group,5%CSE group,Lyn-si RNA+control group,Lyn-si RNA+5%CSE group.The protein expression of E-cadherin,Vimentin,α-SMA,Smad2/3 and p-Smad2/3,Lyn and p-Lyn were detected by Westem blot.In part three,Lyn lentivirus-transfected 16 HBE cells were divided into four treatment groups: GFP+control group,GFP+5%CSE group,Lyn+/++control group,Lyn+/++5%CSE group.The protein expression of E-cadherin,Vimentin,α-SMA,Smad2/3 and p-Smad2/3,Lyn and p-Lyn were detected by Westem blot.Results Part one,the expression of E-cadherin was gradually decreased,and the expression of Vimentin,α-SMA were gradually increased as raising the CSE concentration or prolonging the treated time.When 16 HBE cells were treated with 5%CSE for 72 hours,the E-cadherin expression level reach the lowest value,and the Vimentin,α-SMA expression level reach the peak value.Part two,after Lyn-si RNA-treated: The expression of E-cadherin was decreased,and Vimentin,α-SMA were increased in 5%CSE group compared with the control group,the relative phosphorylation levels of p-Smad2/3 and p-Lyn were increased in 5%CSE group compared with the control group.The expression of E-cadherin was increased,and Vimentin,α-SMA were decreased in Lyn-si RNA+5%CSE group Compared with the 5%CSE group,the relative phosphorylation levels of p-Smad2/3 and p-Lyn were decreased in Lyn-si RNA+5%CSE group Compared with the 5%CSE group.Part three,after Lyn lentivirus-transfected: The expression of E-cadherin was decreased,and Vimentin,α-SMA were increased in GFP+5%CSE group compared with the GFP+control group,the relative phosphorylation levels of p-Smad2/3 and p-Lyn were increased in GFP+5%CSE group compared with the GFP+control group.The expression of E-cadherin was decreased,and Vimentin,α-SMA were increased in Lyn+/++5%CSE group compared with the GFP+5%CSE group,the relative phosphorylation levels of p-Smad2/3 and p-Lyn were increased in Lyn+/++5%CSE group compared with the GFP+5%CSE group.Conclusion As raising the CSE concentration or prolonging the treated time,the EMT was increased in 16 HBE cells,the best stimulus condition was 5%CSE for 72 hours.Lyn-si RNA could inhibition CSE-induced EMT and down-regulation of E-cadherin expression,the underlying mechanism was associated with the decreased relative phosphorylation levels of Smad2/3.overexpression of Lyn kinase could promotion CSE-induced EMT and down-regulation of E-cadherin expression,the underlying mechanism was associated with the increased relative phosphorylation levels of Smad2/3.