| Part Ⅰ Construction and Analysis of a ceRNA Network and Patterns of Immune Infiltration in Chronic Rhinosinusitis with Nasal PolypsObjective: Recent studies have revealed the significant role of the competing endogenous RNA(ce RNA)network in human diseases.However,studies on the mechanism of the role of the ce RNA network in chronic rhinosinusitis with nasal polyps(CRSw NP)are relatively limited.Therefore,this study constructed a ce RNA network by bioinformatics analysis to identify potential regulatory axes in CRSw NP for final experimental validation.Methods: In this study,the GSE136825 dataset from the Gene Expression Omnibus(GEO)was used to investigate differentially expressed lnc RNAs and m RNAs.The GSE169376 dataset was obtained to analyze differentially expressed mi RNAs.Functional analysis was performed via Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)-Gene Set Enrichment Analysis(GSEA).Then,a competitive endogenousRNA(ceRNA)network was constructed based on lnc RNA-mi RNA pairs and mi RNA-m RNA pairs.To validate the key genes lnc RNA and m RNA in the ce RNA axis,GSE36830 and GSE179265 datasets were selected for validation.Based on this,immune cell infiltration in CRSw NP was assessed by CIBERSORT to analyze the correlation between the ce RNA axis(lnc RNA and m RNA)and the infiltrated immune cells.Finally,to validate the key genes,this study was experimentally verified using immunohistochemistry,immunofluorescence staining,and Western blotting.Results: In this study,565 DE-lncRNAs,23 DE-miRNAs,and 1799 DE-m RNAs were identified using the DESeq2 R package and limma R package for CRSw NP patients and controls.Enrichment analysis of 1799 DE-m RNAs showed that CRSw NP was associated with inflammation and immunity.Associations between differentially expressed RNAs were predicted in multiple databases,and 21 lnc RNAs,8 mi RNAs and 8 m RNAs were further identified,and the lncRNA-miRNA-mRNA ce RNA network was constructed by their relationship pairs.A potential MIAT/mi R-125a/IRF4 axis was determined according to the degree and positive correlation between a lnc RNA and its competitive endogenous m RNAs.Based on the results of the GSEA analysis,it can be inferred that IRF4 may be involved in the immune cell infiltration process.Validation of the GSE36830 dataset confirmed that MIAT and IRF4 were differentially expressed between CRSw NP and controls.The area under the ROC curve(AUC)of MIAT and IRF4 was 0.944,suggesting their high accuracy in distinguishing CRSw NP patients from controls.The CIBERSORT analysis revealed that eosinophils and M2 macrophages might be involved in CRSw NP occurrence and development.In addition,MIAT was correlated with dendritic cells and M2 macrophages,and IRF4 was associated with dendritic cells.Finally,the expression of IRF4 in nasal polyps was further confirmed by experimental validation to be significantly higher than that in controls.Conclusions: The constructed novel MIAT/miR-125a/IRF4 axis may play a critical role in the occurrence and development of CRSw NP by bioinformatics analysis.Meanwhile,by constructing ce RNA networks and studying immune cell infiltration,new molecular therapeutic targets for CRSw NP could be identified,providing new therapeutic ideas for the treatment of this disease.Part Ⅱ Inhibition of IL-4/STAT6/IRF4 Signaling Reduces the Epithelial-mesenchymal Transition in Eosinophilic Chronic Rhinosinusitis with Nasal PolypsObjective: Previous studies have shown that epithelial-to-mesenchymal transition(EMT)in nasal epithelial cells is critical for tissue remodeling of chronic rhinosinusitis with nasal polyps(CRSw NP).However,the precise mechanism underlying the EMT remains poorly understood.This study aimed to investigate the role of the Th2 cytokine IL-4 in regulating the EMT process in eosinophilic CRSwNP(Eos CRSwNP).Methods: In this study,quantitative real-time polymerase chain reaction(q RT-PCR),immunohistochemistry,immunofluorescent staining,and Western blotting were performed to evaluate the expression of STAT6,IRF4,and EMT markers in nasal mucosal samples.In addition,the expression of STAT6,IRF4,and EMT markers in h NECs of primary human nasal mucosal epithelial cells(h NECs)from Eos CRSw NP patients after IL-4 stimulation was also examined.Primary hNECs were pretreated with STAT6 inhibitor AS1517499 and then treated with IL-4.EMT and EMT-related markers of primary h NECs,and the regulation of IL-4 on STAT6 and IRF4 were evaluated by wound scratch assay,Western blotting,and immunofluorescence cytochemistry.Results: STAT6 and IRF4 m RNA and protein expression were significantly upregulated in both Eos CRSw NP and Non-Eos CRSw NP compared with control tissues.The expression of STAT6 and IRF4 in Eos CRSw NP was higher than those in Non-Eos CRSw NP(P < 0.05).STAT6 and IRF4 were not only expressed in epithelial cells but also in macrophages.The number of STAT6+CD68+ cells and IRF4+CD68+ cells in Eos CRSwNP were higher than those in Non-EosCRSwNP and control tissues(P < 0.05).E-cadherin and ZO-1 expression was decreased,and N-cadherin and vimentin expression was elevated in Eos CRSw NP compared to control and Non-Eos CRSw NP.IL-4-stimulated primary hNECs exhibited EMT characteristics.Inhibition of STAT6 by AS1517499 inhibits IL-4-induced IRF4 expression and EMT in epithelial cells.Conclusion: STAT6 and IRF4 expression were elevated in Eos CRSw NP.IL-4-induced STAT6 signaling upregulated IRF4 expression in h NECs.IL-4 induced STAT6 upregulation of IRF4 expression in h NECs.IL-4 promotes EMT in h NECs via the STAT6/IRF4 signaling pathway.Part Ⅲ Macrophage M2 Polarization Promotes Epithelial-mesenchymal Transition in Eosinophilic Chronic Sinusitis with Nasal PolypsObjective: Epithelial cells and macrophages are important components of the microenvironment of the nasal mucosa.The interaction between epithelial cells and macrophages regulates the development of various diseases.The purpose of this study was to investigate the interaction between macrophages and epithelial cells to promote EMT in epithelial cells.Methods: In this study,immunohistochemistry,immunofluorescence staining,and Western blotting were used to detect the expression of macrophage markers CD68 and CD163 in nasal mucosal specimens.Differentiation of human THP-1 monocytes into M0 macrophages was performed by stimulation with PMA,followed by stimulation with lipopolysaccharide(LPS)and IFN-γ into M1 macrophages and IL-4 into M2 macrophages.Macrophage phenotype markers and the expression of STST6,IRF4,and MMP9 after treatment of M0 macrophages with IL-4 and AS1517499 were assessed by Western blotting.A co-culture system was also established in this study to explore the interaction between macrophages (THP-1 cells)and h NECs.After co-culture with M2 macrophages,EMT-related markers of primary h NECs were evaluated by immunofluorescence cytochemistry and Western blotting.In addition,we used ELISA to detect TGF-β1 expression in THP-1-derived supernatants.Results: Compared with the control and Non-EosCRSw NP group,the expression of CD68 and CD163 protein in Eos CRSw NP were significantly upregulated.IL-4 stimulates macrophage M2 polarization.The levels of MMP9 and TGF-β1 in M2 macrophages induced by IL-4 stimulation were significantly higher than those in the control group.Inhibition of STAT6 by AS1517499 inhibited IL-4-induced M2 polarization and reduced the levels of IRF4,MMP9,and TGF-β1 in macrophages.Expression of the EMT markers E-cadherin and ZO-1 was reduced in h NECs co-cultured with M2 macrophages(P < 0.05),and vimentin expression showed a slight but non-significant increase(P > 0.05).Conclusion: In this study,we found an increase in M2 macrophages in Eos CRSw NP.Macrophages promote the process of EMT in epithelial cells,and M2 macrophages are involved in the formation process of CRSw NP by enhancing the occurrence of EMT in epithelial cells.We also found that STAT6 inhibitors inhibit the EMT process of epithelial cells by suppressing macrophage polarization toward M2,and this result provides a new strategy for the treatment of nasal polyps. |
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