Mechanism Research Of BRD4 Mediated NF-?B/RelA Signaling Pathway To Regulate The Airway Epithelial-Mesenchymal Transition In Airway Remodeling Of Asthma

With the acceleration of urbanization in the developing countries,the prevalence of asthma increased year by year.The prevalence ranging from 1% to 16% in various countries.The number of patient with asthma worldwide has grown to 300 million including more than 30 million asthma patients in our country.The increased airway resistance and hyper-responsiveness caused by airway remodelling are the main causes of chronic chest tightness and expiratory dyspnea.At present,epithelial to mesenchymal transition(EMT)plays an important role in airway remodeling of asthma,and there are many cytokines and signal pathway involved in the regulation of EMT.Bromodomain 4(BRD4)is a member of the family of bromine structure domain protein,and participates in the process of tumor proliferation,metastasis and inflammation signal transduction.Our preliminary study found that BRD4 mRNA and protein levels were significantly increased in the airways tissues of asthma mice,and BRD4 specific inhibitors JQ1 can inhibit airway remodeling of asthma mice,but the exact mechanism is unclear and needs further studies.Research purpose:Discuss the role and molecular mechanism of BRD4 mediated NF-?B pathway regulating EMT in the process of asthma airway remodeling and provide an theoretical and experimental basis and new therapeutic target for the treatment of asthma and airway remodeling prevention.Experimental methods:Part 1:Explore the regulation mechanism of NF-?B pathway on EMT process of airway epithelium1.After stimulated by TGF?1 72 hours,we observed the phenotypic changes of human bronchial epithelium cell.Then the NF-?B siRNA transfection HBE cells after TGF?1 stimulating,using HE staining to observe the morphologic change of cells andimmunofluorescence to test the change of EMT related transcription factors and the cell phenotype mRNA and protein expression2.Use qRT-PCR and Western blot to detect the changes in the mRNA and protein expression levels of NF-?B,Snail,Vimentin and Ecadherin Part 2:Discuss the role and molecular mechanism of BRD4 mediated NF-?B pathway regulating EMT in the process of asthma airway remodeling.The BRD4 siRNA transfection HBE cells after TGF?1 stimulating,HE staining and cell immunofluorescence experiments to test NF-?B interference that influence EMT related transcription factors and the cell phenotype protein expression;After BRD4 silencing,qRT-PCR and Western blot to detect the changes in the mRNA and protein expression levels of NF-? B,Snail,Vimentin and E-cadherinAfter the treatment of BRD4 specific inhibitor JQ1,HE staining observed cell morphological changes and cell immunofluorescence experiments to detect the changes of cell phenotype protein expression;qRT-PCR and Western blot to detect the cell changes in the expression levels of NF-B,EMT related transcription factors Snail and cell phenotype genes in the mRNA and protein expression levels.Results:1.Clarifies the regulation mechanism of NF-?B pathway on airway epithelial EMT process1.1 The morphology of HBE cells stimulated by TGF?1 is obviously irregular,hypertrophied and elongated into a spindle,Performance as a Spindle-like fibroblasts characteristics,and A large number of free cells.after NF-?B siRNA interference,the cell morphology is more regular and epithelial.The cell morphology is obvious,but some cell morphology still appears fibroblast-like.1.2.After stimulated by TGF?1,the expression of F-actin and Vimentin in stromal cells was significantly increased,and the expression of epithelial phenotype protein E-cadherin was significantly decreased.After interference with NF-?B siRNA,The EMT program of HBE cells was significantly inhibited.1.3 After stimulated by TGF?1,the expression of Snail,Vimentin,NF-?B mRNA and protein were significantly increased,and E-cadherin was significantlydecreased.After treatment with NF-?B siRNA,the expression of Snail,Vimentin,NF-?B mRNA and protein were decreased.,and E-cadherin was increased.2.Discusses the role and molecular mechanism of BRD4 Participate in the process of airway remodeling in asthma through NF-?B pathway.2.1 After the HBE cells stimulated by TGF?1,HBE cells are hypertrophic and elongate into a spindle shape,Performance as a Spindle-like fibroblasts characteristics;on the basis of TGF?1 stimulation,given BRD4 siRNA interference or JQ1 again,most HBE cells have epithelial cell morphology.A small amount of cell morphology was fibroblast-like.2.2 After the HBE cells stimulated by TGF?1,the expression of Fail-actin and stromal cell phenotype proteins Snail and Vimentin was significantly increased in HBE cells,and the expression of E-cadherin in epithelial cells was significantly decreased.On the basis of TGF?1 stimulation,given BRD4 siRNA interference or JQ1 again.The expression of F-actin and Stromal cell phenotype protein Snail and Vimentin were decreased,and the expression of epithelial phenotype protein E-cadherin were increased.2.3 After the HBE cells stimulated by TGF?1,The expression of Snail,Vimentin,NF-?B,BRD4 mRNA and protein were significantly increased,and the expression of E-cadherin was significantly decreased.On the basis of TGF?1 stimulation,given BRD4 siRNA interference or JQ1 again.The expression of Snail,Vimentin,NF-?B mRNA and protein was decreased,and the expression of E-cadherin was increased.Conclusions:In conclusion,our study demonstrated that NF-?B signaling pathway is involved in regulating the EMT program of airway epithelial cells,and BRD4 can significantly inhibit the EMT program of HBE cells by inhibiting the expression of key molecules of NF-?B signaling pathway.This study elucidated the mechanism of NF-?B signaling pathway on EMT in airway epithelial cells,and identified BRD4 as a key target of NF-?B signaling pathway to regulate EMT in airway epithelial cells,providing a new theoretical basis for the treatment of asthma with BRD4 as a target.