Regulation Of Exosomal MiR-129-2-3p On Airway Epithelial Barrier Function In PM_2.5 Aggravated Asthma

Background and Objective:PM_2.5,as an important air pollutant,is closely related to the onset and exacerbation of asthma.Airway epithelial cells are the first physical defense barrier against external allergens and other risk factors,and their damage is often the initiation factor in the development of asthma.Many studies have shown that exosomal miRNAs play a key role in asthmatic airway epithelial barrier dysfunction.The aim of this study was to investigate the mechanisms,possible targets and signaling pathways of exosomal miRNAs in regulating airway epithelial cell barrier function in PM_2.5 aggravated asthma,so as to provide new ideas for elucidating the mechanism of PM_2.5 induced asthma exacerbation and reference for further potential biomarkers and therapeutic targets of asthma.Methods:PartⅠBioinformatics analysis and validation of asthma-related differential miRNAs1.The GEO25230 dataset was selected and analyzed for differentially expressed miRNAs between healthy controls and patients with asthma.The differential miRNAs were visualized using bioinformatics analysis and validated in asthmatic mouse lung tissues,then miR-129-2-3p was selected as the target miRNA.2.The mouse model of asthma was induced by intraperitoneal injection and aerosol inhalation of OVA,then intranasal instillation of PM_2.5 suspension with different concentrations and miR-129-2-3p antagomir/NC.After 40 days,the mice were euthanized and peripheral blood,bronchoalveolar lavage fluid,lung and bronchial tissues were collected.Identification of asthma models:HE and PAS staining were used to observe the pathological changes of lung and bronchial tissues,calculated organ coefficients,detected the level of OVA-specific Ig E in peripheral blood by ELISA,and classified and counted the cells in alveolar lavage fluid.The level of miR-129-2-3p in mouse lung tissue was detected by q RT-PCR.Plasma exosomes were extracted and identified by transmission electron microscopy,nanoparticle tracking analyzer and Western blotting.q RT-PCR was performed to detect the expression levels of miR-129-2-3p in mouse plasma exosomes.3.Fifteen patients with asthma were recruited and matched 1:1 with 15 healthy controls,and then plasma exosomes were extracted.The expression levels of miR-129-2-3p were detected by q RT-PCR,followed by ROC curve analysis.Then correlation analysis was performed between plasma exosomal miR-129-2-3p and PM_2.5 exposure levels.4.The expression level of miR-129-2-3p was detected by q RT-PCR in 16HBE cells stimulated with PBS or 100μg/m L PM_2.5suspension for 48 h.Cell culture supernatants were collected to extract exosomes,and the morphology and size were observed by transmission electron microscopy,particle size was determined by nanoparticle tracking analyzer,and exosome marker proteins were detected by Western blot.After co-culture of exosomes with normal 16HBE cells for 48 h,the uptake of exosomes by 16HBE cells was observed by fluorescence microscopy.The expression level of miR-129-2-3p in exosomes and in 16HBE cells after co-culture with exosomes were detected by q RT-PCR.PartⅡRegulation of airway epithelial barrier function by exosomal miR-129-2-3p1.Western blotting and q RT-PCR were used to detect the levels of ligand proteins ZO-1,occludin,E-cadherin and inflammatory factors IL-6,CXCL15,TNF-αin lung tissues of PM_2.5 aggravated asthmatic mice;ELISA was used to detect the levels of inflammatory factors IL-6,CXCL15 and TNF-αin BALF.Immunohistochemistry was used to observe the localization and expression changes of ZO-1,occludin and E-cadherin in lung tissues.2.After stimulating 16HBE cells with PBS or 100μg/m L PM_2.5 suspension for 48 h,respectively,the permeability alteration of 16HBE cells was evaluated by detecting the permeability of FITC-dextrose;Western blotting and q RT-PCR were used to detect the cellular ligand proteins ZO-1,occludin,E-cadherin and the levels of inflammatory factors IL-6,IL-8 and TNF-α.3.After transfecting 16HBE cells with miR-129-2-3p mimics/inhibitor for 48 h,the permeability of 16HBE cells was evaluated by detecting the permeability of FITC-dextrose;q RT-PCR was used to detect the levels of miR-129-2-3p in cells;Western blotting and q RT-PCR were used to detect the levels of the ligand proteins ZO-1,occludin,E-cadherin and inflammatory factors IL-6,IL-8 and TNF-αin cells.4.After co-culture of exosomes with normal 16HBE cells for 48 h,the permeability of 16HBE cells was evaluated by detecting the permeability of FITC-dextrose;Western blotting and q RT-PCR were used to detect the levels of the ligand proteins ZO-1,occludin,E-cadherin and inflammatory factors IL-6,IL-8 and TNF-αin cells.5.16HBE cells transfected with miR-129-2-3p mimics/inhibitor were then co-cultured with Exo-PM_2.5 for 48 h,the permeability of 16HBE cells was evaluated by detecting the permeability of FITC-dextrose;Western blotting and q RT-PCR were used to detect the levels of the ligand proteins ZO-1,occludin,E-cadherin and inflammatory factors IL-6,IL-8 and TNF-αin cells.PartⅢExosomal miR-129-2-3p regulates airway epithelial barrier function through targeted inhibition of TIAM1/RAC1/PAK1 signaling pathway1.Three target prediction software programs,Target Scan,miRDB,and miRWalk were used to predict putative targets of miR-129-2-3p.Venn diagrams,gene ontology（GO）functional enrichment analysis and KEGG pathway enrichment were used to identify target genes and major biological pathways.The dual-luciferase reporter system showed that TIAM1 was target gene of the miR-129-2-3p.2.In the PM_2.5 exacerbated asthma mouse model,the levels of signaling pathway molecules TIAM1,RAC1,p-RAC1,PAK1 and p-PAK1 were detected by Western blotting and q RT-PCR;the localization and expression of TIAM1 in lung tissues were observed by immunohistochemistry.3.16HBE cells were stimulated with PM_2.5,transfected with miR-129-2-3p mimics/inhibitors,co-cultured with exosomes from cell supernatants,or transfected with miR-129-2-3p mimics/inhibitors and then co-cultured with Exo-PM_2.5.Western blot and q RT-PCR were then used to detect the expression levels of signaling pathway molecules TIAM1,RAC1,p-RAC1,PAK1 and p-PAK1.4.After transfecting 16HBE cells with siR-TIAM1/NC,siR-TIAM1/NC+miR-129-2-3p-inhibitor for 48 h,respectively,the permeability of epithelial cells was evaluated by detecting the permeability of FITC-dextrose.Western blotting and q RT-PCR were used to detect the expression levels of signaling pathway molecules TIAM1,RAC1,p-RAC1,PAK1,p-PAK1;ligand proteins ZO-1,occludin,E-cadherin and inflammatory factors IL-6,IL-8,TNF-α.Results:PartⅠBioinformatics analysis and validation of asthma-related differential miRNAs1.A total of 13 differentially expressed miRNAs were screened from the GEO25230dataset,including 11 up-regulated miRNAs and 2 down-regulated miRNAs.miR-129-2-3p was selected as the target miRNA after verification.2.A mouse model of PM_2.5exacerbated asthma was successfully established:HE and PAS staining showed obvious inflammatory cell infiltration and goblet cell hyperplasia in lung and trachea tissues of the OVA group,inflammatory cell infiltration gradually increased,goblet cell hyperplasia aggravated,mucus secretion enhanced with increasing PM_2.5exposure dose.The plasma levels of OVA-specific Ig E in OVA and PM_2.5 groups were significantly higher than those in NS group.The lung coefficient in OVA group was remarkably raised than that in NS group,and raised in PM_2.5 group in a dose-response relationship.The total number of cells,percentage of neutrophils,eosinophils and lymphocytes in each PM_2.5 group were notably increased,while the percentage of macrophages was significantly decreased.The levels of miR-129-2-3p in lung tissues were significantly higher in the OVA group compared with the NS group.In the PM_2.5-exposed group,the levels of miR-129-2-3p in lung tissues gradually increased with the increase of PM_2.5 poisoning concentration.After miR-129-2-3p antagomir intervention,the levels of miR-129-2-3p in lung tissues were significantly decreased.The plasma exosomes of mice were extracted and identified to be consistent with the basic characteristics of exosomes.The expression level of exosomal miR-129-2-3p was significantly increased in the OVA group and gradually increased with the increase of PM_2.5 poisoning concentration.After miR-129-2-3p antagomir intervention,the expression level of plasma exosomal miR-129-2-3p in mice was significantly reduced.3.The level of plasma exosomal miR-129-2-3p was significantly higher in the patient with asthma group than that in the healthy control group.ROC curve analysis revealed that exosomal miR-129-2-3p had a high ability to discriminate between patients with asthma and healthy controls.Exosomal miR-129-2-3p was significantly and positively correlated with PM_2.5 exposure levels.4.PM_2.5 exposure resulted in elevated miR-129-2-3p expression levels in 16HBE cells.Exosomes in the culture supernatant were extracted and identified as consistent with the characteristics of exosomes.miR-129-2-3p levels were significantly higher in PM_2.5-induced exosomes(Exo-PM_2.5)than in PBS-induced exosomes（Exo-NC）.Co-culture of exosomes with normal 16HBE cells revealed that exosomes could be successfully taken up by 16HBE cells,and the expression level of miR-129-2-3p was significantly enhanced in 16HBE cells co-cultured with Exo-PM_2.5.PartⅡRegulation of airway epithelial barrier function by exosomal miR-129-2-3p1.In the PM_2.5 exacerbated asthma mouse model,the m RNA and protein levels of the ligand proteins ZO-1,occludin,and E-cadherin were remarkably lower in the lung tissue of the OVA group than in the NS group,while the m RNA levels of the inflammatory factors IL-6,Cxcl15,and Tnfαwere vastly improved in the OVA group.The levels of ZO-1,occludin and E-cadherin decreased gradually with the increase of PM_2.5 dose,while the levels of IL-6,Cxcl15 and Tnfαincreased sharply.After miR-129-2-3p antagomir intervention,asthma symptoms were relieved,and the m RNA and protein levels of ZO-1,occludin and E-cadherin were significantly higher than those in the HPM_2.5 group,while the m RNA levels of the inflammatory factors IL-6,Cxcl15 and Tnfαwere obviously lower than those in the HPM_2.5 group.Immunohistochemical results showed that ZO-1,occludin and E-cadherin were expressed in bronchial epithelial cells of lung tissues,and the expression levels reduced progressively with increasing poisoning dose of PM_2.5,but raised markedly after miR-129-2-3p antagomir intervention.2.PM_2.5 stimulated 16HBE cells,resulting in decreased expression of the ligand proteins ZO-1,occludin and E-cadherin in the cells,elevated cell permeability,and enhanced release of the inflammatory cytokines IL-6,IL-8 and TNF-α.3.Transfection of miR-129-2-3p mimics could substantially increase the expression of miR-129-2-3p in 16HBE cells,thereby reducing the expression of ZO-1,occludin and E-cadherin,enhancing cell permeability,and promoting the release of inflammatory factors.However,transfection of miR-129-2-3p inhibitor could diminish the level of miR-129-2-3p in cells,promote the expression of ZO-1,occludin and E-cadherin,weaken cell permeability and inhibit the release of inflammatory factors.4.Exo-PM_2.5 reduced the m RNA and protein levels of the ligand proteins ZO-1,occludin and E-cadherin in 16HBE cells,enhanced 16HBE cell permeability,and promoted the release of the inflammatory factors IL-6,IL-8 and TNF-α.5.16HBE cells transfected with miR-129-2-3p mimics then co-cultured with Exo-PM_2.5 further reduced the expression of connexin in the cells and enhanced cell permeability and inflammatory factor release.In contrast,transfection with miR-129-2-3p inhibitor followed by co-culture with Exo-PM_2.5 antagonized the effects induced by Exo-PM_2.5.PartⅢExosomal miR-129-2-3p regulates airway epithelial barrier function through targeted inhibition of TIAM1/RAC1/PAK1 signaling pathway1.A total of 195 target genes of miR-129-2-3p were predicted.The target gene TIAM1was selected by GO functional annotation and KEGG pathway enrichment analysis.The dual-luciferase reporter system verified that TIAM1 was the target gene of miR-129-2-3p.2.In the PM_2.5exacerbated asthma mouse model,the expression levels of signaling pathway molecules TIAM1,RAC1,p-RAC1,PAK1,and p-PAK1 in lung tissues of the OVA group were sharply lower than those of the NS group;the expression levels of signaling pathway molecules gradually decreased with the increase of PM_2.5 poisoning dose.After miR-129-2-3p antagomir intervention,the expression levels of signaling pathway molecules were dramatically higher than those in the HPM_2.5 group.3.PM_2.5 stimulation of 16HBE cells markedly reduced the expression levels of signaling pathway molecules TIAM1,RAC1,p-RAC1,PAK1,and p-PAK1 in the cells.Exo-PM_2.5 co-culture with 16HBE cells inhibited the TIAM1/RAC1/PAK1 signaling pathway.Transfection of miR-129-2-3p mimics significantly alleviated the expression levels of signaling pathway molecules TIAM1,RAC1,p-RAC1,PAK1,and p-PAK1 in 16HBE cells;while transfection of miR-129-2-3p inhibitor promoted the expression of signaling pathway molecules.16HBE cells transfected with miR-129-2-3p mimics and then co-cultured with Exo-PM_2.5 further reduced the expression of signaling pathway molecules in the cells,while transfection with miR-129-2-3p inhibitor and then co-cultured with Exo-PM_2.5 effectively antagonized the effect caused by Exo-PM_2.5.4.Inhibition of TIAM1 with siRNA could memorably enhanced 16HBE cell permeability,decreased the expression levels of signaling pathway molecules TIAM1,RAC1,p-RAC1,PAK1,p-PAK1 and the ligand proteins ZO-1,ocludin,E-cadherin,and promoted the release of inflammatory factors IL-6,IL-8,and TNF-α.If 16HBE cells transfected with siR-TIAM1 were then intervened with miR-129-2-3p-inhibitor,it was shown that miR-129-2-3p-inhibitor could antagonize the effect caused by siR-TIAM1.Conclusion:1.Mi R-129-2-3p was significantly elevated in plasma exosomes from PM_2.5exacerbated asthma mouse model,plasma exosomes from patients with asthma,and exosomes released from 16HBE cells stimulated by PM_2.5,which can be used as a potential biomarker for asthma diagnosis.2.Exosomal miR-129-2-3p could exacerbate PM_2.5-induced impairment of epithelial cell barrier function.3.Exosomal miR-129-2-3p impaired airway epithelial barrier function and promoted inflammatory responses by targeting inhibition of the TIAM1/RAC1/PAK1 signaling pathway.Inhibition of miR-129-2-3p could repair airway epithelial barrier function and reduce the inflammatory response.This suggested that exosomal miR-129-2-3p can be used as a target for asthma treatment.