The Role And Mechanism Of MiR-221-3p In Airway Eosinophil Inflammation In Asthma

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Correlation between airway epithelial cells,induced sputum and plasma miR-221-3p expression and airway eosinophil inflammation in asthmatic patientsObjective: To detect the expression of miR-221-3p in airway brushings,sputum and plasma in asthmatic and healthy subjects,and to explore the correlation between airway inflammation and the severity of airway eosinophils.The changes in the expression of miR-221-3p in the airway epithelial cells and induced sputum in the patients with asthma were detected before and after the treatment with inhaled hormone.Method: We recruited 77 asthma patients,36 cases of healthy subjects to examine for airway brushings,induced sputum and miR-221-3p in plasma,and in situ hybridization was used to locate miR-221-3p in the cytoplasm of airway epithelium.The correlation between the expression of miR-221-3p and the proportion of eosinophils in the sputum,the number of eosinophils in the biopsy area,exhaled NO,peripheral blood eosinophil volume and plasma total Ig E were analyzed.We detected the expression of marker genes in Th2 inflammation such as CLCA1,POSTN and SERPINB2 by real-time PCR,and analyzed the correlation between them and the miR-221-3p of airway brushings in patients with asthma.Lung function and provocation test as one of the diagnostic criteria for asthma was performed and analyzed its correlation with miR-221-3p of airway brushings,further the correlation between miR-221-3p and airway eosinophil inflammation was confirmed by detection of induced sputum miR-221-3p before and after ICS treatment.Results: There was no significant difference in age and sex ratio between the two groups of the healthy control group and the bronchial asthma group.Since the proportion of eosinophils in the sputum,the number of eosinophils in the biopsy area,the eosinophil volume in peripheral blood and the total plasma Ig E in the bronchial asthma group were significantly higher than those in the healthy control group.While the FEV1% value of the bronchial asthma group and the PD20 value of the bronchial provocation test were markly lower than those in the healthy control group.Airway biopsy specimens detected by in situ hybridization showed that miR-221-3p mainly expressed the cytoplasm of airway epithelial cells,and miR-221-3p expression in asthmatic group was considerably lower than that in healthy controls.The miR-221-3p transcriptional level in the airway brushings,induced sputum and plasma in the bronchial asthma group was significantly lower than that in the healthy control group.We found that there were significant negative correlations between miR-221-3p in bronchial asthma airway brushings and sputum eosinophil percentage(r=-0.70,p<0.001),the number of eosinophils in the biopsy area(r=-0.61,p<0.001),Fe NO(r=-0.69,p<0.001),the amount of peripheral blood eosinophils(r=-0.59,p<0.001),total plasma Ig E(r=-0.42,p<0.001),marker genes of Th2(r=-0.69,p<0.001),but with the prediction value of FEV1%(r=0.40,p=0.02)and bronchial provocation test PD20 values(r= 0.67,p<0.001)there were significantly positively relations.The transcription level of miR-221-3p in induced sputum and peripheral blood of asthma group were significantly lower than that of control group.Mi R-221-3p in induced sputum or plasma had positively relation with miR-221-3p in airway brushings,and showed significant negative correlations with the inflammation indexes.Mi R-221-3p in induced sputum of before and after the treatment of ICS was detected.After the treatment of ICS,the decreased miR-221-3p transcriptional level was partly recovered.Conclusion: Mi R-221-3p was significantly decreased in airway brushings,induced sputum and plasma in bronchial asthma patients,and mainly expressed in the cytoplasmic parts of the airway epithelial cells.There is a significant negative correlation between the miR-221-3p transcriptional level and the severity of eosinophil inflammation in bronchial asthma patients.The correlation between miR-221-3p in induced sputum and plasma and miR-221-3p in airway brushings was a significant positive,also had significant negative correlation with the degree of airway eosinophil inflammation.The expression level of miR-221-3p in induced sputum was changed before and after ICS treatment,and the miR-221-3p transcriptional level in airway epithelial cells was restored to some extent after treatment.Part ? CXCL17,a target of miR-221-3p,inhibits the expression of inflammatory cytokines via p38 MAPK signaling pathway in airway epithelial cellsObjective: To determine CXCL17 is the target gene of miR-221-3p,and to explore the mechanism of CXCL17 in airway inflammation in asthma.The effects of dexamethasone on the transcriptional level of miR-221-3p and CXCL17 were observed.Method: We used Th2 cytokine IL-13 to stimulate BEAS-2B cells.BEAS-2B cells transfected with miR-221-3p mimic and inhibitor were used to measure the changes of airway inflammatory factors in asthma by real-time PCR.We used multiple biological calculation and analysis software to screen the target gene of miR-221-3p.Double fluorescence test was used to verify that CXCL17 is target gene of miR-221-3p.The expression and location of CXCL17 in the airway epithelium were detected by real-time PCR and IHC,the correlation with the expression of miR-221-3p in the airway brushings was analyzed.The changes of CXCL17 after using dexamethasone was detected by real-time PCR method,and the expression levels of CCL24,CCL26 and POSTN after transfected with CXCL17 si RNA and overexpression plasmid or stimulated by p38 MAPK inhibitor SB 203580 were determined by real-time PCR and ELISA.Results: IL-13 stimulates BEAS-2B cells,miR-221-3p in cells decreases at the transcriptional level.At the same time,the expression of inflammatory factors,CCL24,CCL26,and POSTN is increased.The transfection of miR-221-3p inhibitor caused decrease of transcription level and protein level of CCL24,CCL26 and POSTN.The transcriptional level and protein level of CCL24,CCL26 and POSTN were increased by transfection of miR-221-3p mimic.Dexamethasone recovered the reduce of miR-221-3p caused by IL-13 stimulation,and relieved the increase of inflammatory factors.The double fluorescence experiment shows that CXCL17 is the target gene of miR-221-3p,and there is a combination of the two pairs in the base reverse complementary pair.The expression of CXCL17 in the airway epithelium of asthma group increased identified by real-time PCR and immunohistochemistry at the same time.There was a significant negative correlation between CXCL17 expression and the expression of miR-221-3p in airway brushings.Dexamethasone restores the increase of CXCL17 caused by IL-13 stimulation.By transfected with CXCL17 si RNA and overexpression plasmids or stimulated by p38 MAPK inhibitor SB 203580,there are significant changes in transcriptional level and protein expression level of CCL24,CCL26 and POSTN,which testified that CXCL17 regulates CCL24,CCL26 and POSTN by P38 MAPK signaling pathway.Conclusion: miR-221-3p plays an important role in the expression of inflammatory cytokines CCL24,CCL26 and POSTN in the airway epithelial cells.CXCL17 is the target gene of miR-221-3p.Dexamethasone recovered the decrease of miR-221-3p,and alleviated the increase of CXCL17 induced by IL-13 stimulation.The expression of CXCL17 was increased in the cytoplasm of the airway epithelium in asthmatic patients,and had significant negative correlation with the expression of miR-221-3p in the airway brushings.CXCL17 regulates the CCL24,CCL26 and POSTN stimulated by IL-13 through the p38 MAPK signaling pathway.Part ? Airway overexpression of miR-221-3p aggravates the airway inflammation and mucus secretion in the model of HDM airway inflammation in miceObjective: To explore the expression of miR-221-3p in the mice asthma model of HDM and its role in airway inflammation and mucus secretion.Method: We constructed mice HDM model of asthma,and detected the expression of miR-221-3p,Il-4,Il-5,Il-13,Ccl24,Ccl26 and Postn in the lung tissues of mice by real-time PCR.The extent of infiltration was graded according to the HE staining of paraffin section in the lung tissue of mice.The number of cells in the alveolar lavage fluid(BALF)and the count of macrophages,eosinophils,neutrophils and lymphocytes were counted.ELISA was used to estimate the total Ig E in mice plasma,as well as the changes of Il-4,Il-5,Il-13,Ccl24 and Postn in protein levels in BALF.In addition,the transcriptional level of the mucous expression gene Muc5 ac and Muc5 b in lung tissues of mice was detected real-time PCR and airway mucus expression was evaluated by glycogen staining.Results: In mice HDM model of asthma the decrease expression of miR-221-3p in the cytoplasm of the airway epithelial cells.Airway overexpression of miR-221-3p in mice increased airway inflammation,which caused increase infiltration of eosinophils and other inflammatory cells around the airway,also increased the amount of total number of BALF cells,macrophages,especially eosinophils.The total plasma Ig E and Il-4,Il-5,Il-13,Ccl24,Ccl26,Postn in the lung tissue were increased by the airway overexpression of miR-221-3p.In addition,airway overexpression of miR-221-3p raised airway mucus secretion.Conclusion: In the asthma model of HDM,the expression of miR-221-3p in the mouse's lung tissue decreased.The airway overexpression of miR-221-3p aggravated the airway inflammation and increased the secretion of airway mucus in mice.