Lyn Regulates The Expression Of MUC5AC In Bronchial Epithelial Cells By TGF-?/SMAD

objective:Bronchial asthma(asthma)is an important health issue facing the world,its morbidity and mortality are rising year by year.At present,airway mucus hyper-secretion in severe asthma leading to airway obstruction,lung function decline,increasing infection is the most important problem.The current study of airway mucus hyper-secretion in asthma focus on the inflammatory mediators an their relative cellular signal molecular mechanisms How Lyn regulating TGF-?/SMAD cellular signal transduction in the asthmatic airway mucus hyper-secretion is rarely reported.Therefore our studies mainly focus on this molecular mechanism.Methods:(1)Construct Lyn hyper-expression vector by gateway technology and Package Lyn hyper-expression lentivirus by liposome transfection 293T cells.(2)Construct the human MUC5AC promoter Luciferase Report Gene(pGL3-hMUC5AC/-689/+48)fragment according to our laboratory construction of human MUC5AC promoter Luciferase Report Gene(pGL3-hMUC5AC/-1300/+48).(3)Cell experimental groups:First of all,Lyn hyper-expression virus transfecting human bronchial epithelial cells and non Lyn hyper-expression viral transfection,and then respectively with TGF-?(1,2,3)stimulating human bronchial epithelial cells by virus infection,PBS stimulation group for control;One another,Lyn siRNA interfering human bronchial epithelial cells group and non transfection of Lyn siRNA in human bronchial epithelial cells group,and then respectively stimulating transfected human bronchial epithelial cells with TGF-?(1,2,3),PBS stimulation group for control.(4)Detecting MUC5AC expression,respectively,by immuno-fluorescence and Promega's dual luciferase report gene;testing the expression of ?-actin,Smad2/3,pSmad2/3 and Lyn by western blot;Elisa was used to detect the expression of TGF-?(1,2,3)in Lyn hyper-expression transgenic mice's serum with OVA sensitizing.Results:1.it was successful for lentvirus packaging and vector construction.2.Human MUC5AC promoter Luciferase Report Gene(pGL3-hMUC5AC/-689/+48)fragments were successfully constructed,and sequence alignment,and expected the identical nucleotide sequence.3.TGF-?1 and TGF-?3,respectively,stimulating wild type(non interference of LynsiRNA or non transfection of Lyn high expression virus)human bronchial epithelial cells increased MUC5AC protein expression and MUC5AC gene activation compared with the PBS control group(P<0.05),the gray value of Smad2/3,pSmad2/3 also increased by western blot testing(P<0.05),but TGF-?2 stimulation of wild type human bronchial epithelial cells did not increase the expression of MUC5AC protein and the activity of MUC5AC gene compared with PBS control group(P>0.05),and detection the gray value of Smad2/3,pSmad2/3 by western blot compared the control group had no significance difference(P>0.05).4.TGF-?2 and TGF-?3 respectively stimulating the deletion of Lyn(the group of LynsiRNA interference)in human bronchial epithelial cells promoted MUC5AC protein expression and active MUC5AC gene promoter which was significantly higher than that of the wild type(P<0.05),and Western blot detection of Smad2/3 and pSmad2/3 gray value increased significantly(P<0.05),but TGF-?1 stimulus human bronchial epithelial cells lack of Lyn greatly decreased the expression of MUC5AC protein and the activity of MUC5AC promoter(P<0.05),and significantly decreased the gray value of Smad2/3,pSmad2/3 detection by western blot(P<0.05).5.TGF-?2 and TGF-?3 stimulation Lyn up-regulated(the transfection of Lyn high expression virus)human bronchial epithelial,leading to the expression of MUC5AC protein and the activity of MUC5AC gene promoter decreasing which was significantly lower than that of the wild type(P<0.05),also the gray value of Smad2/3,pSmad2/3(p<0.05),but the expression of MUC5AC protein and the activity of MUC5AC gene were not reduced by TGF-?1 stimulation(P>0.05),and the expression of Smad2/3,pSmad2/3 had no statistically signigicant differences in TGF-?1 inducing Lyn up-regulated human bronchial cells comparing with wild type(P>0.05).Conclusions:1.TGF-?1 and TGF-?3 increase the expression of MUC5AC in human bronchial epithelial,TGF-?2 did not promote the expression of MUC5AC in human bronchial epithelial.2.After deletion of Lyn,TGF-?2 and TGF-?3 could significantly increase MUC5AC expression of human bronchial epithelial cells,and TGF-P 1 could significantly reducethe expression of MUC5AC in human bronchial epithelial cells.3.Because of up-regulation of Lyn,TGF-?2 and TGF-?3 can reduce the MUC5AC expression of human bronchial epithelial cells,but TGF-P 1 cannot reduce the expression of MUC5AC in human bronchial epithelial cells.4.The molecular mechanism of Lyn regulation the MUC5AC expression in bronchial epithelial cells in TGF-?stimulus may be ralated to the expression of Smad2/3 and pSmad2/3,and There is correlation between the increase or decrease of the MUC5AC expression and the up-regulation or down regulation of the Smad2/3 and pSmad2/3 expression.