Study On The Regulatory Mechanism Of MiRNA-9/Th-17/Treg In Chronic Sinusitis

Background and objective:Chronic Rhinosinusitis(CRS)as an upper respiratory diseases,are prevalent worldwide.During the disease development,persistent and severe inflammatory responses is one main characteristic,which affect quality of life.However,there are still few methods to relieve the symptom,because the key immune molecules and regulatory mechanisms in the pathological process of CRS are still unclear.T-cell immune responses have been shown to play an important role in many inflammatory diseases of the upper respirator,including CRS.In T-cell immunity,Th17 cell subgroup,as a unique lineage,is characterized by the secretion of IL-17A.Recent studies have shown that Th17 may also play an important role in the pathophysiology of upper respiratory diseases.Th17 cells can promote the aggregation of eosinophils and neutrophils in CRS.In addition,some evidence suggests that the Th17 response is also regulated by endogenous and exogenous components in the pathological process of CRS.Nasal mucosal epithelial cells are the main cell type involved in the construction of nasal microenvironment.Nasal epithelial cells express almost all toll-like receptor(TLR),especially TLR2,TLR4 and TLR5.Activated by these stimuli,epithelial cells can not only express chemokines and cytokines,but also produce many recognition molecules to mediate subsequent immune responses.Nasal polyps which are another characteristic of CRS is believed to be generated through the process of epithelial mesenchymal transformation,in which epithelial cells are also involved.MicroRNAs are short single-strand RNA molecules.They are able to downregulate or silence the gene expression by inhibiting the transcription or translation of mRNAs.Dysregulation of miRNA expression has been reported in a variety of diseases,including cancer,inflammatory skin and bowel diseases,rheumatoid arthritis and asthma.Studies on miRNAs in upper respiratory diseases indicated that miRNAs were involved in the inflammation of the upper respiratory.There have also been a large number of reports that miRNAs were involved in the regulation process of Th17 immune response.In this study,we study the role of IL-17A in the inflammatory process of CRS,and to explore the regulatory mechanism of miRNA-9.Research contents and methods:1.Nasal mucosal tissues were collected and stored at-80?for further molecular experiments.Chronic sinusitis pathological samples for pathological analysis were detected by HE staining,Masson staining,Tunnel staining,alcian blue staining and immunohistochemical detection.2.Target specific analysis of miRNAs:miRNAs and target were confirmed according to the prediction of target gene of miR-9 by bioinformatics.Wild-type/mutant plasmids were constructed and co-transfected with miR-9 mimics,and the targeted regulatory effects of the two plasmids were detected by dual-luciferase assay.3.Analysis of the regulatory mechanism of miRNA-9/IL-17A in vitro experiment:expression of miRNA-9/IL-17A/FOXP3 in vitro;the transcription levels of miRNA-9,IL-17A and FOXP3 were analysis by qPCR.Effects of different miR-9 levels on miRNA-9/IL-17A/FOXP3 in vitro;Co-effects of miR-9 and IL-17A in vitro;Results:Patients with chronic sinusitis(CRSwNP)showed significant Th17 immune response and suppressed Treg immune response,and the level of miR-9 in nasal mucosal tissues showed a significant negative correlation with the IL-17A.According to bioinformatics,the binding site of miR-9 and target gene IL17A was predicted.The mutant plasmid IL-17A was developed and for miR-9 target-specific analysis.Compared with the control group,miR-9 significantly inhibited the expression of luciferin reporter gene of wild-type IL-17A plasmid but not the mutant one,suggesting that the prediction of binding sites was correct and specific.Further,HNEpC,a cell line of nasal mucosa epithelial cells was cultured and stimulated with LPS as a CRS inflammatory model.The results showed miR-9 and FOXP3 transcription levels were negative relative with the LPS level,but IL-17A transcription level was positive relative with the LPS level.Expression of FOXP3 and IL-17A showed a positive correlation with each transcription level.The expression of FOXP3 gradually decreased with the LPS concentration increasing,while the expression of IL-17A gradually increased.Further,miR-9 inhibitors were added to analyze the effects of miR-9 on IL-17A and FOXP3.The transcriptional expression of miR-9,IL-17A,FOXP3,MMP-9,E-cadherin and N-cadherin were analyzed.Results showed that the transcriptional expression levels of miR-9 and FOXP3 were significantly decreased in the miR-9 inhibitor group,and the transcriptional expression levels of IL-17A and N-cadherin were significantly increased.The transcriptional expression levels of E-cadherin were decreased in the miR-9 inhibitor group,while the transcription level of MMP-9 was increased.The immunofluorescence results of E-cadherin,IL-17A and N-cadherin were consistent with western blot's result.Further,when IL-17A was overexpressed,the transcription levels of FOXP3 and E-cadherin were significantly down-regulated,but the transcription levels of MMP-9 and N-cadherin were significantly up-regulated.Meanwhile,the expression levels of Wnt3a were also significantly up-regulated.Conclusion:According to the above results,in the process of CRS,there is a significant Th17 inflammation,which leads to a distinct inflammatory and epithelial mesenchymal transformation in the nasal cavity,and miR-9 is involved in this process.IL-17A is regulated by miR-9,and the epithelial mesenchymal transformation of nasal mucosa epithelial cells is inhibited.This suggests that miR-9/IL-17A pathway plays an important role in the pathological process of chronic sinusitis.