| Background and Objective:Allergic rhinitis(AR)is a Th2-type inflammatory reaction of the nasal mucosa mediated by Immunoglobulin E(Ig E)after atopic individuals are exposed to allergens.Clinically,AR is characterized by nasal itching,sneezing,runny nose and nasal congestion.The recurrent occurrence of AR can induce some common complications,such as asthma,nasal polyps and sinusitis,etc.At present,the treatment of AR includes symptomatic treatment represented by steroid hormones and antihistamines and cause-based treatment represented by specific immunotherapy,but the above treatment methods have varying degrees of limitations,and many patients are still not satisfied with the efficacy.Therefore,it is of great scientific value and potential clinical significance to explore the molecular mechanism of AR occurrence so as to accurately target key molecular targets for intervention.Group 2 innate lymphoid cells(ILC2s)mainly exist in the mucosal barrier.The nasal epithelium of specific individuals will produce epithelial cytokines to stimulate the activation of ILC2 s after being stimulated by allergens.After activation,ILC2 s can produce a large number of Th2 cytokines(IL-4,IL-5 and IL-13)in the early stage of inflammation,and participate in allergic inflammatory response earlier and stronger than adaptive immunity.The previous study of our group found that the expression of miR-155-5p was increased in the nasal mucosa of AR patients,which was positively correlated with the proportion of ILC2 s,and could also affect the content of Th2 cytokines.It is suggested that miR-155-5p may be involved in regulating the function of ILC2 s in AR,but the specific mechanism is not yet clear.This study intends to identify the existence of ETS1-miR-155-5p-TLE4 molecular regulatory axis in AR,and to explore the biological function of this regulatory axis on ILC2 s in the allergic environment and its specific role in the occurrence of AR.Methods:(1)To explore the expression of ETS1 in AR and its effect on ILC2s:Bioinformatics analysis of miR-155-5p transcription factors;JASPAR database predicts the binding site of the miR-155 host gene(MIR155HG)promoter and ETS1;From September 2019 to June 2021,the data of 71 patients(37 cases in AR group and34 cases in control group)in the Department of Otolaryngology Head and Neck Surgery of the Second Affiliated Hospital of Nanchang University were collected.Mucosal tissue and peripheral blood were collected during the operation.The expression of ETS1 in nasal mucosal tissue was detected by Quantitative real time polymerase chain reaction(q RT-PCR)and Western blotting(WB);the proportion of ILC2 s was detected by flow cytometry;the correlation between the expression of ETS1 and the the proportion of ILC2 s was statistically analyzed.The AR mouse model was constructed.After intranasal treatment with Lv-sh ETS1,the expression of ETS1 and its effects on nasal symptoms and pathological changes were observed by behavioral scoring,nasal mucosa HE staining,q RT-PCR and WB;the proportion of ILC2 s of mice was detected by flow cytometry;The content of Th2 cytokine(IL-4,IL-5 and IL-13)in serum of mice was detected by enzyme-linked immunosorbent assay(ELISA).ILC2 s derived from AR mice were transfected with Lv-ETS1 or Lv-sh ETS1,the proliferation and apoptosis of ILC2 s were observed by CCK8,Ed U and flow cytometry;The content of Th2 cytokines(IL-4,IL-5 and IL-13)in supernatant was determined by ELISA.(2)To explorethe regulatory relationship between ETS1 and miR-155-5p in AR:The expression of miR-155-5p in clinical samples was detected by q RT-PCR;the correlation between the expression of miR-155-5p,the proportion of ILC2 s and the expression of ETS1 was statistically analyzed.The expression of miR-155-5p in mice was detected by q RT-PCR.ILC2 s derived from AR mice were transfected with Lv-ETS1 or Lv-sh ETS1,and the expression of miR-155-5p in each group was determined by q RT-PCR;the dual-luciferase gene reporter assay and Chromatin Immunoprecipitation(Ch IP)to verify the regulatory relationship between ETS1 and miR-155-5p;ILC2s derived from AR mice were co-transfected with Lv-ETS1 and anti-miR155-5p,the proliferation and apoptosis of ILC2 s were observed by CCK8,Ed U and flow cytometry;The content of Th2 cytokines(IL-4,IL-5 and IL-13)in supernatant was determined by ELISA.(3)To explore the molecular mechanism of the ETS1-miR-155-5p axis in regulating ILC2 s in AR: The target gene prediction of miR-155-5p was performed using the miRNAs target gene prediction database(Target Scan,Pictar,miRanda,PITA and miRmap),and the differential expression gene data of activated and non-activated ILC2 s were screened through the GEO database to find the significantly down-regulated target genes in activated ILC2s;the expression of TLE4 in clinical samples was detected by q RT-PCR and WB.The correlation between the expression of TLE4 and ILC2 s,miR-155-5p and TLE4 was statistically analyzed;Target Scan database to find the target binding sequence of miR-155-5p and TLE4;the dual luciferase assay was used to verify the regulatory relationship between miR-155-5p and TLE4;The expression of TLE4 in the nasal mucosa of mice was detected by q RT-PCR and WB;ILC2s derived from AR mice were co-transfected with Lv-ETS1 and anti-miR155-5p,and the expression of TLE4 in each group was determined by q RT-PCR.ILC2 s derived from AR mice were transfected with Lv-sh ETS1 or Lv-sh TLE4,the proliferation and apoptosis of ILC2 s were observed by CCK8,Ed U and flow cytometry;The content of Th2 cytokines(IL-4,IL-5 and IL-13)in supernatant was determined by ELISA.Results:(1)Bioinformatics analysis showed that ETS1 Bioinformatics analysis found that ETS1 is an upstream transcription factor of miR-155-5p;JASPAR database predicts the binding site of the MIR155 HG promoter and ETS1.The proportion of ILC2 s in the peripheral blood of AR patients was significantly higher than that of the control group(P <0.05).ETS1 was high expressed in AR,and its expression was positively correlated with the proportion of ILC2s(P <0.05).AR mouse model experiment showed that compared with the NC group,the expression of ETS1 in the nasal mucosa of the AR group was increased(P <0.05),nasal symptoms were obvious,the nasal mucosa was pathologically changed,the proportion of ILC2 s in vivo and the contents of Th2 cytokines(IL-4,IL-5 and IL-13)in serum were increased(P <0.05);The expression of ETS1 in nasal mucosa of mice was significantly inhibited after intranasal treatment with Lv-sh ETS1(P <0.05),compared with Lv-sh Con group,nasal symptoms of the Lv-sh ETS1 group were partially relieved,and the pathological changes of the nasal mucosa were alleviated,the proportion of ILC2 s in vivo and the content of IL-4 IL-5 and IL-13 in serum were decreased(P <0.05).In vitro cell experiments showed that compared with Lv-Con group,the proliferation ability of ILC2 s increased,the apoptosis ratio decreased,and the concentrations of Th2 cytokines(IL-4,IL-5 and IL-13)in the cell supernatant increased after ETS1 overexpression(P <0.05);Compared with Lv-sh Con group,the proliferation ability of ILC2 s decreased,the apoptosis ratio increased,and the concentrations of Th2 cytokines(IL-4,IL-5 and IL-13)in the cell supernatant decreased after ETS1 interference(P <0.05).(2)miR-155-5p was high expressed in AR,and its expression was positively correlated with ETS1 and the proportion of ILC2s(P <0.05).AR mouse model experiment showed that compared with the NC group,the expression of miR-155-5p in nasal mucosa tissues of mice in AR group was increased,and the expression of miR-155-5p could be inhibited by nasal instillation of Lv-sh ETS1 in mice(P <0.05).In vitro cell experiments showed that,the expression of miR-155-5p was significantly increased after ETS1 overexpression,while the expression of miR-155-5p decreased after ETS1 knockdown(P <0.05).Dual luciferase gene reporter assay and Ch IP assay showed that ETS1 as a transcription factor could bind to the predicted binding site of miR-155-5p.The rescue experiments showed that the increased proliferation ability,decreased apoptosis ratio and increased Th2 cytokines(IL-4,IL-5 and IL-13)concentration in cell supernatant induced by overexpression of ETS1 could be partially reversed by miR-155-5p downregulation(P <0.05).(3)Take the intersection of ILC2 s differentially expressed genes and miR-155-5p target gene prediction results to obtain candidate target genes TLE4;TLE4 was low expressed in AR,and its expression was negatively correlated with miR-155-5p,ETS1 and the proportion of ILC2s(P <0.05).Dual-luciferase gene reporter assays verify that miR-155-5p targets TLE4 3′-UTR.AR mouse model experiment showed that compared with the NC group,the expression of TLE4 in nasal mucosa tissues of mice in AR group was decreased,and the expression of TLE4 could be inhibited by nasal instillation of Lv-sh ETS1 in mice(P <0.05);The rescue experiments showed that knockdown of miR-155-5p up-regulate the expression of TLE4 in ILC2 s,and partially reverse the elevated expression of TLE4 caused by overexpression of ETS1.Transfection of Lv-sh TLE4 or Lv-sh ETS1 into ILC2 s showed that the decreased proliferation ability,increased apoptosis ratio and decreased Th2 cytokines(IL-4,IL-5 and IL-13)concentration in cell supernatant induced by ETS1 knockdown could be partially reversed by TLE4 downregulation(P<0.05).Conclusions:(1)ETS1 was highly expressed in AR,and its expression level is positively correlated with the proportion of ILC2 s and the expression of miR-155-5p,but negatively correlated with the expression of TLE4.(2)The expression of ETS1 was closely related to the nasal symptoms,histopathological structure of nasal mucosa,the proportion of ILC2 s in vivo and the concentration of Th2 cytokines(IL-4,IL-5 and IL-13)in the serum of AR mice.(3)ETS1 regulates the expression of TLE4 by mediating the transcription of miR-155-5p,thereby affecting the proliferation,activation and apoptosis of ILC2 s in AR. |
PDF Full Text Request