| Background:Allergic Rhinitis(AR)is a common nasal mucosa allergic disease characterized by the rise of serum-specific IgE and the increase in type II cytokines in the nasal mucosa.Recent studies have shown that Group 2 Innate Lymphoid cells(ILC2s)play a role in AR.ILC2 s are newly discovered innate immune cells that do not express any antigenspecific receptors and have similar functions of TH2 cells.ILC2 s regulate the immune response by releasing type 2 cytokines such as IL-4,IL-5,and IL-13 in response to epithelial-derived IL-33 and IL-25 after antigen exposure.However,the mechanism by which ILC2 s regulate their cytokines secretion in AR is still not well understood.MiR-155 is a functional non-coding RNA closely related to immune function.MiR-155 has been shown to be involved in the development of allergic diseases by regulating a variety of immune cell functions.We and others have recently demonstrated that miR-155 plays an essential role in the function of ILC2 s in allergic airway inflammation models such as AR.However,whether miR-155 directly regulates ILC2 s function in vitro and through which regulatory mechanisms have not been well studied.Therefore,the purpose of this study is to verify whether miR-155 regulates ILC2 s function in vitro and explore its regulatory mechanism.Method:1.ILC2 s were isolated from peripheral blood mononuclear cells(PBMC)by immunomagnetic bead sorting and cultured in vitro.Immunofluorescence staining and flow cytometry were used to detect the isolation purity of ILC2 s.2.MiR-155 mimics or inhibitor were transfected to ILC2 s for up-regulating or inhibiting miR-155 expression respectively.After stimulating with IL-33 and IL-25,RT-qPCR and ELISA were used to detect the expression and secretion of IL-4,IL-5 and IL-13 in ILC2.3.The miR-155 target genes were predicted from three databases: miRDB,TargetScan,and miRTarBase.The target genes that may be involved in ILC2 s function were screened out based on the ILC2 s chip data in the GEO database and verified by RT-qPCR.Double luciferase assay was performed to verify the binding site of miR-155 to TP53INP1 3’UTR.4.To assess whether TP53INP1 overexpression alters the regulation of miR-155 on secretion of IL-4,IL-5,and IL-13 by ILC2 s,the cells were co-transfected with miR-155 mimics and TP53INP1 overexpression lentiviral vector.After stimulating with IL-33 and IL-25,ELISA was used to detect IL-4,IL-5 and IL-13 in ILC2 s supernatant.result:1.The results of fluorescence microscopy showed that the proportion of ILC2 s in PBMC was 0.18%(0.18 ± 0.016%)before sorting and 73.7%(73.70 ± 2.17%)after magnetic beads sorting.Similarly,flow cytometry showed that the proportion of ILC2 s in PBMC increased from 4.69%(4.69 ± 0.16%)before sorting to 70.94%(70.94 ± 0.071%).2.Overexpression of miR-155 significantly increased the expression of IL-4 and IL-5 mRNA in ILC2 s while down-regulating miR-155 obviously inhibited the expression of these cytokines.However,no significant changes were found in IL-5 mRNA expression.Furthermore,overexpression of miR-155 was showed to elevate the concentration of IL-4,IL-5,and IL-13 in ILC2 s culture supernatant.On the contrary,down-regulating miR-155 inhibited the concentration of these cytokines.3.Through target gene prediction,we obtained 110 target genes of miR-155.Combining with the chip data from GEO datebase,we found 9 of these target genes might be involved in ILC2 s function.RT-qPCR results showed that among these functional target genes,overexpression of miR-155 inhibited the expression of HBP1,CREBRF,TLE4,TP53INP1,KBTDB2 mRNA in ILC2 s.Finally,the binding site of miR-155 and TP53INP1 3’UTR was comfirmed by double luciferase assay.4.Compared with the control group,overexpression of TP53INP1 partially inhibited the secretion of IL-4 and IL-13 that induced by overexpression of miR-155,but no significant change in IL-5 secretion was found.Conclusion:MiR-155 regulates ILC2 s function in AR by targeting TP53INP1 3’UTR. |
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