Brg1 Inhibits E-cadherin Expression In Lung Epithelial Cells And Disrupts Epithelial Integrity

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ABNORMAL EPRESSION OF BRG1 IN LUNG TISSUE AND AIRWAY EPITHELIUM OF ASTHMATIC C57BL/6 MICEOBJECTIVE: To observe the abnormal expression of Brg1 gene in lung tissue and airway epithelium of asthmatic C57BL/6 mice;to investigate the correlation between Brg1 and asthma.METHODS: The mice were divided into two groups: normal control group?control?and asthma group?asthma?.Mice were detected by AHR,bronchoalveolar lavage and BALF cell count,while sediment smear of BALF cells were used for cell counting and classification within 48 hours after the final challenge of OVA.The pulmonary pathological tissue were used for hematoxylin-eosin?HE?staining and anti-E-cadherin immunohistochemical staining.Lung tissue specimens were taken for m RNA and Real-time PCR protein and WB detection.RESULTS: The m RNA and protein levels of Brg1 from lung tissue were significantly increased in asthma group.Anti-Brg1 immunohistochemical staining showed that the increased expression of Brg1 in airway epithelium of asthmatic group.CONCLUSION: The m RNA and protein levels of Brg1 in asthmatic mice were significantly increased,suggesting that Brg1 has some correlation with asthma.Meanwhile,the overexpression of Brg1 by airway epithelium in asthma group indicated a certain relationship between Brg1 and airway epithelial.Study of Brg1 maybe helpful for further research of other epithelial repair mechanisms in asthma.Part ? EFFECTS OF BRG1 KNOCKOUT ON AIRWAY EPITHELIAL INTEGRITY IN C57BL/6 ASTHMITIC MICEOBJECTIVE: To observe the effect of Brg1 on AHR,airway inflammation and epithelial marker E-cadherin in asthmatic mice.METHODS: Cre recombinase transgenic mice?SFTPC-rt TA/?Tet O?7?and lox P transgenic heterozygous mice?Brg1lox P/lox P?were screened for homozygous knockout mice(Brg1^-/-)after hybridization.The asthma mice were divided into four groups: wild type control group?WT CTRL?,wild type mice with asthma?WT asthma?and Brg1 knockout mice control group(Brg1^-/-Ctrl),Brg1 knockout mice with asthma(Brg1^-/-asthma).Mice were detected by AHR,bronchoalveolar lavage and BALF cell count,while sediment smear of BALF cells were used for cell counting and classification within 48 hours after the final challenge of OVA.The pulmonary pathological tissue were used for hematoxylin-eosin?HE?staining and antiE-cadherin immunohistochemical staining.Lung tissue specimens were taken for m RNA and Real-time PCR protein and WB detection.RESULTS: The results revealed a noticeable reduction in Brg1 expression by bronchial epithelium and lung tissue in Brg1^-/-mice.We found that LR in Brg1^-/-asthmatic mice was significantly lower than that in WT asthmatic mice.The number of total cells and eosinophils in bronchoalveolar lavage fluid?BALF?in the Brg1^-/-group was also significantly reduced.In addition,the number of inflammatory cells around the bronchi was much lower.IHC revealed that E-cadherin expression by the bronchial epithelium in WT asthmatic mice was lower than that in WT controls.Meanwhile,the distribution of E-cadherin was significantly larger in Brg1^-/-asthmatic mice than in WT asthmatic mice.Moreover,WB analysis revealed that expression of E-cadherin in lung tissue from Brg1^-/-asthmatic mice was higher than that in WT asthmatic mice,which was consistent with the results of IHC.CONCLUSION: These phenomena suggest that knocking down Brg1 improves lung function by increasing AHR,reducing inflammatory cell infiltration around bronchi,and reducing mucus secretion by goblet cells,suggesting that Brg1 knockout could enhance the integrity of the asthma epithelium.Part ? STUDY ON THE MECHANISM OF THE EFFECT OF BRG1 KNOCKDOWN ON THE REPAIR ABILITY OF HUMAN BRONCHIAL EPITHELIAL CELL LINE 16HBEOBJECTIVE: To investigate the effect and mechanism of Brg1 on proliferation and migration of human bronchial epithelial cells?16HBE?after lentivirus Brg1-sh RNA transfection.METHODS: Lentivirus Brg1 expression vector and packaging plasmids?packaging Mix?were used to transfect into 293 T cells after construction of lentiviral Brg1 interference vector.Then,virus were packaged,packaged lentivirus?Brg1-sh RNA?and negative control lentivirus?Brg1-sc RNA?were used to infect 16 HBE.Western Blot and q PCR were used to verify the stable transfection.The migration and proliferation ability of two cell lines were detected by cell scratch assay,transwell chamber and CCK8,flow cytometry.The E-cadherin expression were detected by IHC and WB,and the other two epithelial markers ZO-1 and Occlubin1 were detected by Q-PCR.Double luciferase reporter gene analysis was used to examin the interaction between Brg1 and E-cadherin promoter region in two groups of cells.The binding strength of Brg1 and E-cadherin promoter region of the two groups of cells was detected by Ch IP-PCR.RESULTS: Cell scratch test and Transwell assay showed that Brg1 knockdown could promote the migration of airway epithelial cells.CCK8 assay and flow cytometry showed that Brg1 knockdown could promote the airway epithelial cell proliferation,as cell proliferation mainly manifested as increased the S phase of the cell cycle.Q-PCR and WB results showed increased E-cadherin m RNA and protein level in Brg1-sh RNA cell line compared with non transfected 16 HBE cells and Brg1-sc RNA cell lines.Meanwhile,no obvious change of ZO-1 and Occlubin1 m RNA level were observed.Dual-luciferase reporter assays revealed that Brg1 interacted with a-1964/+941 bp segment of the E-cadherin promoter in cells exposed to Brg1 sc RNA but not in cells exposed to Brg1 sh RNA.After that,we used Ch IP-PCR to identify the bound sequences in 16 HBE cells and we found that Brg1 interacted most strongly with the sequence-86/+60.CONCLUSION: Knockdown of Brg1 could enhance the migration and proliferation of 16 HBE cells,mainly because through Brg1 inhibits transcriptional activation of E-cadherin by binding to the E-cadherin promoter.