The Expression And Molecular Mechanism Of AGR3 In COPD Patients With Frequent Exacerbator

Objectives: chronic obstructive pulmonary disease(COPD)is a complex and heterogeneous disease.According to the different mechanisms appear in the different patients,COPD has different clinical prognosis including symptoms,acute exacerbation,response to treatment,disease progression speed or death.Therefore,COPD is divided into different phenotypes according to the attributes of individual differences.Compared with infrequent acute exacerbation phenotype,patients with frequent acute exacerbation phenotype have poor quality of life,increased mortality,decreased lung function and poor prognosis.Thus,frequent acute exacerbation phenotype in COPD is more meanful of our attention.However,the research on the pathogenesis of frequent acute exacerbations of COPD is still in its infancy,lacking effective methods for early identification and prevention.Therefore,based on the results of frequent acute exacerbation phenotype proteomics,this study further explored the mechanism of the role of AGR3 in the occurrence and development of frequent acute exacerbation phenotype of COPD through cell experiments in vitro.Methods:Part Ⅰ: expression of AGR3 in the phenotype of COPD with frequent acute exacerbations1)Immunofluorescence and immunohistochemistry were used to determine the location of AGR3 expression in human lung tissue and the differences in the expression of AGR3 in human lung tissue among control group,infrequent acute exacerbation of COPD phenotype group(IFCOPD)and frequent acute exacerbation of COPD phenotype group(FCOPD).2)Western blot was used to detect the expression of protein in lung tissues of control group,infrequent acute exacerbation of COPD and frequent acute exacerbation of COPD.3)RT-PCR was used to detect the expression of m RNA in lung tissues of control group,COPD infrequent acute exacerbation phenotype group and COPD frequent acute exacerbation phenotype group.4)ELISA was used to detect the presence of AGR3 in the serum of patients with frequent acute exacerbation phenotype,and to compare the differences among control group,infrequent acute exacerbation phenotype group and frequent acute exacerbation phenotype group.The accuracy,specificity and sensitivity of the biomarker model were analyzed by ROC curve.Part Ⅱ: the role of AGR3 in epithelial barrier function of COPD with frequent acute exacerbation phenotype through regulating tight junction protein and adhesion junction protein1)Immunohistochemistry was used to determine the expression of occludin,ZO-1 and E-cadherin in bronchial epithelial cells of lung tissue in control group,infrequent acute exacerbation of COPD phenotype group and frequent acute exacerbation of COPD phenotype group.2)The expression of occludin,ZO-1 and E-cadherin was measured in cigarette smoking extract(CSE)-exposed BEAS-2B cells model.3)AGR3 over expressed and slince BEAS-2B cells model was made through lentivirus and DNA plasmid transfection.The expression of occludin,ZO-1 and E-cadherin was measured in AGR3 over expressed and slince BEAS-2B cells exposed by CSE.Part Ⅲ: AGR3 participates in airway inflammation of epithelial cells with frequent acute exacerbation phenotype of COPD by regulating E-cadherin / β-catenin complex and Wnt / β-catenin signaling pathway1)The expression of E-cadherin and β-Catenin in lung tissues of control group,non-frequent acute exacerbation of COPD and frequent acute exacerbation of COPD were determined by immunohistochemistry.2)The expression of E-cadherin and β-Catenin in CSE-exposed BEAS-2B cells model was detected by Western blot and RT-PCR.3)The expression of E-cadherin and β-Catenin was measured in AGR3 over expressed and slince BEAS-2B cells exposed by CSE.Observe the effect of AGR3 in E-cadherin /β-catenin complex.Results:Part Ⅰ: expression of AGR3 in the phenotype of COPD with frequent acute exacerbations1)The expression of AGR3 in bronchial epithelial cells of human lung tissue was determined by immunofluorescence and immunohistochemistry.It was found that the expression of AGR3 in bronchial epithelial cells of human lung tissue with frequent acute exacerbation phenotype(FCOPD)was lower than that with infrequent acute exacerbation phenotype(IFCOPD).2)Western blot showed that the expression of AGR3 in the lung tissue of frequent acute exacerbation phenotype patients was lower than that of infrequent acute exacerbation phenotype patients.3)RT-PCR showed that the expression of AGR3 m RNA in the lung tissue of frequent acute exacerbation phenotype patients was lower than that of infrequent acute exacerbation phenotype patients.4)The expression of AGR3 in human serum samples with frequent acute exacerbation phenotype was lower than that with infrequent acute exacerbation phenotype.ROC curve analysis showed that the accuracy,specificity and sensitivity of AGR3 biomarker model were high.Part Ⅱ: the role of AGR3 in epithelial barrier function of COPD with frequent acute exacerbation phenotype through regulating tight junction protein and adhesion junction protein1)The expression of E-cadherin,occludin and ZO-1 was detected by immunohistochemistry.Compared with infrequent acute exacerbation phenotype group,the expression of E-cadherin,occludin and ZO-1 was decreased in frequent acute exacerbation phenotype group.2)The results showed that more than 5% concentration of CSE could significantly inhibit BEAS-2B cell viability,and the higher the concentration was,the more obvious the inhibition was.Western blot and RT-PCR were used to confirm that the protein and m RNA levels of E-cadherin,occludin and ZO-1 were significantly decreased in CSE-exposed BEAS-2B cells model stimulated by 10% cigarette extract.3)The overexpression of AGR3 in BEAS-2B cells increased the expression of E-cadherin,occludin and ZO-1,while the expression of E-cadherin,occludin and ZO-1decreased in the silence of AGR3 in BEAS-2B cells.The results showed that the overexpression of AGR3 could alleviate the decrease of E-cadherin,occludin and ZO-1expression induced by 10% CSE.Part Ⅲ: AGR3 participates in airway inflammation of epithelial cells with frequent acute exacerbation phenotype of COPD by regulating E-cadherin / β-catenin complex1)The expression of E-cadherin and β-catenin in the airway epithelium of COPD patients with frequent acute exacerbation phenotype was detected by immunohistochemistry.2)Western blot and RT-PCR were used to confirm that the protein levels of E-cadherin and β-catenin were significantly decreased in 10% CSE exposed group,and the m RNA level of β-catenin began to decrease in 5% CSE exposed group.3)The results showed that the protein expression of E-cadherin and β-catenin was increased in the AGR3 overexpression BEAS-2B cells,while the protein expression of E-cadherin and β-catenin was decreased in the AGR3 silencing BEAS-2B cells.The interaction between E-cadherin and β-catenin was enhanced in the overexpression of AGR3 in BEAS-2B cells by CO immunoprecipitation.Conclusion:1.The expression of AGR3 in bronchial epithelial cells of human lung tissue decreased in both protein and m RNA levels in lung tissue of patients with frequent acute exacerbation phenotype.AGR3 was expressed in serum samples of patients,and the expression level decreased.The accuracy,specificity and sensitivity of AGR3 biomarker model are high,and it still needs to be further verified by large serum samples.2.The expression of E-cadherin,occludin,ZO-1 in the airway epithelium of patients with frequent acute exacerbation of COPD was lower than that of patients with infrequent acute exacerbation of COPD.The protein and m RNA levels of E-cadherin,occludin and ZO-1 were significantly decreased in the COPD model stimulated by 10%cigarette smoking extract.The expression of E-cadherin,occludin and ZO-1 was increased in AGR3 overexpressed BEAS-2B cells,while the expression of E-cadherin,occludin and ZO-1 was decreased in AGR3 silenced BEAS-2B cells.Overexpression of AGR3 alleviated the decrease of E-cadherin,occludin and ZO-1 expression induced by10% CSE.3.Compared with infrequent acute exacerbation,the expression of E-cadherin and β-catenin in the airway epithelium of the frequent acute exacerbation group was decreased,and the expression of β-catenin was increased in BEAS-2B cells with AGR3 overexpression,while the expression of β-catenin was decreased in BEAS-2B cells with AGR3 silencing.The overexpression of AGR3 mainly regulates the interaction between E-cadherin and β-catenin by enhancing E-cadherin.