Function And Mechanism Of Mir-493-5p Targeting FOXO1 To Regulate The Polarization Of Th9 Cells In Asthma

Background:Bronchial asthma(abbreviated as asthma)is the most common chronic airway inflammatory disease among children.In recent years,the incidence and mortality of asthma have gradually increased worldwide,which poses a great threat to human health and aggravates financial stress on families and society.Asthma has become a global public health problem that needs to be solved urgently.The key is to find intervention measures from the pathogenesis level of asthma.The pathogenesis of asthma is extremely complex.At present,there is no definite conclusion on the specific pathogenesis of asthma.The inhibition of helper T cell type 1(Th1)and the hyperfunction of helper T cell type 2(Th2)lead to the imbalance of Th1/Th2,which is currently considered as the main cause of asthma.However,the theory of Thl/Th2 imbalance cannot explain all the experimental phenomena,because asthma may involve more other cell populations.With the discovery of Th9 cells,a new subgroup of CD4-Th cells,more and more studies have begun to pay attention to the role of these cells in the immunopathology of asthma.Th9 cells are differentiated from Naive CD4-T cells stimulated by IL-4 and TGF-β,and secrete a large amount of IL-9.Th9 cells secreting IL-9 play an important role in the immunopathology of asthma.In recent years,microRNAs(miRNAs),which was initially regarded as transcriptome "noise",has been paid more and more attention by researchers.MiRNA is a non-coding RNA with a length of 18-25 nucleotides,which is highly conserved in the process of evolution.By binding to the 3’Untranslated Regions(3’UTR)of mRNA,it inhibits gene translation and then reduces the expression of target genes,and its role in the pathogenesis of many diseases has been widely studied.There is more and more evidence that miRNAs is involved in the regulation of airway inflammation in asthma.In this study,high-throughput sequencing combined with GO analysis,KEGG signal pathway analysis,informatics analysis and published database was used to screen out miRNAs differentially expressed between asthma group and control group,the purpose of this study is to explore the effect of miR-493-5p on the differentiation of Th9 cells in asthma.Part Ⅰ:Expression and clinical significance of miR-493-5p,FOXO1 and IL-9 in peripheral blood mononuclear cells of children with asthmaObjective:To investigate the expression of miR-493-5p,FOXO1 and IL-9 in peripheral blood mononuclear cells of children with asthma and their correlation with clinical data and related laboratory tests.Methods:1.This study included 30 Chinese children with asthma hospitalized in the Department of Respiratory Medicine,Children’s Hospital of Soochow University as well as 32 Chinese nonasthmatic control children with selective operation hospitalized in Children’s Hospital of Soochow University from September 2017 to June 2019.The diagnosis of asthma were conducted according to the Guidelines for the Diagnosis,Prevention and Treatment of Children’s Bronchial Asthma.At the same time,the clinical data and related laboratory tests were collected,including gender,age,the proportion of eosinophils in peripheral blood of asthmatic children,total IgE level in peripheral blood,FEV1%,FeNO level,the length of inpatient days and so on.2.Collect the peripheral blood samples of asthma group and control group,and screen out differentially expressed miRNAs by high-throughput sequencing combined with GO analysis,KEGG signal pathway analysis,informatics analysis and published database.3.Detect the expression of serum miR-493-5p,FOXO1 and IL-9 in the two groups by real-time quantitative PCR(qRT-PCR)and analyze their correlation;analyze the correlation between the expression of miR-493-5p in peripheral blood and clinical data in asthma group.Results:1.High-throughput sequencing and verification showed that miR-493-5p in peripheral blood of children with asthma decreased significantly than that of healthy controls and may be related to Th differentiation.2.The relative expression of miR-493-5p in peripheral blood of children with asthma was significantly lower than that of healthy controls(P<0.05),and the relative expression of FOXO1,IL-4 and IL-9 in peripheral blood of children with asthma was significantly higher than that of healthy controls(P<0.05).3.There were significant negative correlations between miR-493-5p in asthma group and FOXO1(r=-0.519,P<0.05),IL-9(r=-0.684,P<0.01),IL-4(r=-0.589,P<0.01),the proportion of eosinophils in peripheral blood(r=-0.671,P<0.01),total IgE level in peripheral blood(r=-0.631,P<0.01),FEV1%(r=-0.827,P<0.01)and FeNO level(r=-0.726,P<0.01)respectively;but there were no relations between miR-493-5p in asthma group and the age(r=0.180,P>0.05)or the length of inpatient days(r=0.117,P>0.05).Conclusion:The expression level of miR-493-5p in children with asthma was decreased,and it was negatively correlated with FOXO1,IL-9,IL-4,allergic inflammation index in peripheral blood,FEV1%and FeNO.Part Ⅱ:The role and mechanism of gradual regulation of miR-493-5p/FOXO1/Th9 cellsObjective:To predict and verify that the target gene of miR-493-5p is FOXO1,and to explore the mechanism of miR-493-5p regulating the differentiation of Th9 cells by targeting FOXO1.Methods:1.MiR-493-5p mimic and miR-493-5p inhibitor were transfected into Naive CD4+T cells of wild type mice.After 7 days of Th9-induced differentiation medium culture,the expression level of IL-9 was detected by flow cytometry and ELISA.The mRNA level and protein level of miR-493-5p,FOXO1,IRF4 and IL-9 were detected by qRT-PCR and Western Blot.2.The biological information of miR-493-5p and its possible target gene were analyzed by TargetScan,starBase v2.0 and other software.And FOXO1 was verified to be the target gene of miR-493-5p by double luciferase reporter gene experiment.3.FOXO1 overexpression plasmids and FOXO1 small interference plasmids were constructed and transfected into Na(?)ve CD4+T cells respectively in Th9 induced differentiation culture system.The effect of miR-493-5p on Th9 cell differentiation was further verified by rescue experiment through targeted action on FOXO1.Results:1.The overexpression of miR-493-5p in Naive CD4+T cells could inhibit the expression of FOXO1,IL-9 and IRF4 at the mRNA level and protein level,while silencing miR-493-5p could increase the mRNA and protein levels of FOXO1,IL-9 and IRF4.These suggest that overexpression or silencing of miR-493-5p could inhibit or promote the differentiation of Th9 cells.2.The prediction results of TargetScan,starBase v2.0 and other software show that FOXO1 is a possible direct target gene of miR-493-5p.The results of double luciferase show that the luciferase activity of FOXO1 3’UTR wild type vector co-transfected with miR-493-5p mimic was significantly lower than that of the control group,but the mutant vector had no significant change,suggesting that the target gene of miR-493-5p was FOXO1.3.The design of the rescue experiment was achieved by co-transfection.It was verified that overexpression of FOXO1 could save the differentiation of Th9 cells inhibited by miR-493-5p at mRNA level and protein level.Conclusion:miR-493-5p inhibits the differentiation of Th9 cells and the production of IL-9 by targeting FOXO1,suggesting that miR-493-5p may be a potential target for the treatment of asthma.Part Ⅲ:Role of overexpression of miR-493-5p in an OVA-sensitized asthma modelObjective:To construct a mouse model of asthma sensitized by OVA,achieve the overexpression of miR-493-5p in vivo by the intervention of miR-493-5p agomiR,and explore the role of miR-493-5p in regulating Th9 cell differentiation in the pathogenesis of asthma.Methods:1.6-8 weeks female BALB/c mice were randomly divided into 4 groups:Control group,Asthma group,miR-493-5p agomiR group and agomiR NC group,with 8 mice in each group.2.Establishment of OVA asthma model:on day 0 and 7,100μl OVA sensitization mixture was injected intraperitoneally.OVA atomization challenge was performed on days 17,18,19 and 20.On day 21,lung tissue,BALF and plasma were collected for subsequent detection.miR-493-5p agomiR group was given 40μl of miR-493-5p agomiR via nasal drops on days 14,15 and 16.3.24 hours after the last challenge of OVA,the airway hyperresponsiveness of mice in each group to different concentrations of methacholine was measured by non-invasive pulmonary function meter.The infiltration of inflammatory cells around the airway was evaluated by HE staining,airway inflammation score and BALF cell count.The proliferation of bronchial goblet cells and the secretion of airway mucus was evaluated by PAS staining.The lung tissue and BALF of mice were collected to detect the level of IL-9 by flow cytometry and ELISA,and the mRNA and protein levels of FOXO1,IRF4 and IL-9 in lung tissue were detected by qRT-PCR and Western blot.Results:1.The behavioral score of OVA asthma group was significantly higher than that of control group.After airway transfection of miR-493-5p agomiR,the behavior score of OVA asthma mice was significantly lower than that of OVA asthma group.It is suggested that the overexpression of miR-493-5p in the airway of asthmatic mice can alleviate thebehavioral symptoms of asthmatic mice.2.With the increase of methacholine concentration,the airway responsiveness of OVA asthmatic mice gradually increased,but the airway hyperresponsiveness decreased after airway overexpression of miR-493-5p,which is very beneficial to improve airway symptoms and has a protective effect.3.After overexpression of miR-493-5p in airway of asthmatic mice,inflammatory cell infiltration,goblet cell proliferation,airway mucus secretion and airway inflammation score decreased.After OVA asthmatic mice were transfected with miR-493-5p agomiR,the total cell count,eosinophil percentage and lymphocyte percentage in BALF were lower than those in OVA asthmatic mice,suggesting that miR-493-5p has a protective effect on airway inflammation in OVA asthmatic mice.4.The expression of miR-493-5p in lung tissue of asthmatic mice significantly increased after transfection of miR-493-5p agomiR,suggesting that the transfection of miR-493-5p agomiR was successful.The mRNA and protein levels of FOXO1,IRF4 and IL-9 decreased in varying degrees after overexpression of miR-493-5p in airway.5.The results of flow cytometry showed that the percentage of IL-9+ cells in lung tissue and BALF of asthmatic mice after overexpression of miR-493-5p in airway was lower than that in asthma group.ELISA results showed that the expression of IL-9 in lung tissue and BALF decreased after overexpression of miR-493-5p in airway of asthmatic mice.Conclusion:miR-493-5p inhibits Th9 cell differentiation and alleviates airway inflammation in OVA asthmatic models.miR-493-5p is expected to be a potential target for the treatment of asthma,which provides some evidence for future treatment of asthma.Part Ⅳ:The effect of DCs secreted exosome miR-493-5p on Th9 cell differentiation was preliminarily exploredObjective:From the perspective of exosome miRNAs,to explore the effect of DCs secreted exosome miR-493-5p on Th9 cell differentiation.Methods:1.Bone marrow-derived dendritic cells(BMDCs)were induced from bone marrow of OVA-induced asthma mice and control mice.qRT-PCR was used to detect the expression of miR-493-5p in BMDCs exosomes.2.Dendritic Cells(DCs)were induced from bone marrow of wild type mice.After cultured,the supernatant of DCs was collected and exosomes were extracted by supercentrifugation method.Exosomes-miR-493-5p NC and exosomes-miR-493-5p inhibitor were constructed to transfect into Naive CD4+T cells and cultured in Th9 cell induced differentiation culture system.After 72 hours,the cells were collected and the percentage of IL-9+cells was detected by flow cytometry.Results:1.The expression of miR-493-5p from DCs exosomes in OVA asthmatic mice was significantly lower than that in the control group.2.Naive CD4+T cells transfected with DC exosome miR-493-5p inhibitor in Th9 cell differentiation culture system could increase the expression of IL-9.Conclusion:Exosome miR-493-5p derived from DC inhibits the differentiation of Th9 cells.