Mechanism Of Lnc-CEBPB-13 Regulating Th2 Differentiation In Allergic Rhinitis

Objective:The prevalence of allergic rhinitis(AR)in adults is as high as 10-30%,and about40%of them will develop into asthma,which brings a huge economic burden to society.The pathogenesis of AR is complex,and various treatment methods have certain limitations.Therefore,in-depth research on the mechanism of AR is of great significance.The differentiation imbalance of CD4~+T cells resulted in the occurrence and development of allergic rhinitis(AR).In recent years,the regulatory role of long non-coding RNA(lnc RNA)in immune cell differentiation has attracted more and more attention.Previous studies have found that lnc-CEBPB-13 is significantly overexpressed in the blood of patients with allergic rhinitis,but whether it can regulate helper T(Th)cell differentiation remains unclear.A large number of studies have shown that lnc RNA can bind to micro RNA(mi RNA)sites as competing endogenous RNA(ce RNA)to regulate the expression of m RNA and target genes.Ce RNA play an important role in the genesis and progression of inflammation.The purpose of this study was to investigate the mechanism of lnc-CEBPB-13 regulating GATA binding protein 3(GATA3)through mi-10b-5p and thus regulating Th2 cell differentiation in AR.Methods:A total of 20 peripheral blood samples were collected from AR patients and healthy controls in Yantai Yuhuangding Hospital from January 2020 to June 2022.Peripheral blood mononuclear cells(PBMCs)were extracted for culture.All subjects underwent allergen detection.Spearman correlation analysis was performed between the relative expression level of lnc-CEBPB-13 and allergy grade in subjects.CD4~+T cells were sorted from cultured PBMCs by magnetic beads,and the localization of lnc-CEBPB-13 in CD4~+T cells was detected.The cells were subjected to nuclear and cytoplasmic separation,and RNA was extracted for nucleic acid electrophoresis to detect the intracellular sublocalization of lnc-CEBPB-13.After transfection of lnc-CEBPB-13 overexpression or knockdown in PBMCs,RNA,protein and cell supe RNAtant were extracted to detect the overexpression efficiency of lnc-CEBPB-13,and the expression of Th2 transcription factor GATA3 and inflammatory factors Interleukin(IL)-4/IL-5/IL-13 in AR group and control group.The proportion of Th2 cells in CD4~+T cells after lnc-CEBPB-13 overexpression was analyzed by flow cytometry.The mi RNA regulating Th2 transcription factors with lnc-CEBPB-13 were detected by gene chip technology at the transcriptome level.After overexpression or knockdown of mi R-10b-5p in PBMCs,the expression of Th2 transcription factor GATA3and inflammatory factors IL-4/IL-5/IL-13 were detected,and the proportion of Th2 cells in CD4~+T cells after overexpression of mi R10b-5 was analyzed by flow cytometry.The binding sites of mi R-10b-5p to lnc-CEBPB-13 and GATA3-3’UTR were identified,and the interaction mode of mi R-10b-5p with lnc RNA and GATA3 was detected by dual luciferase assay.The relative expression levels of GATA3 and IL-4/IL-5/IL-13 were detected in PBMCs by simultaneous overexpression of mi R-10b-5p and lnc-CEBPB-13.Results:lnc-CEBPB-13 is highly expressed in AR patients and significantly correlated with the level of allergy.lnc-CEBPB-13 was enriched in CD4+T cells and expressed both inside and outside the nucleus.Overexpression of lnc-CEBPB-13 in PBMCs increased the expression of Th2-related transcription factor GATA3 and inflammatory factors IL-4/IL-5/IL-13,and increased the proportion of Th2 cells in CD4+T cells.Transcriptome sequencing analysis showed that 34 mi RNA including mi R-10b-5p could regulate Th2transcription factors after overexpression of lnc-CEBPB-13.Overexpression or knockdown of lnc-CEBPB-13 caused the opposite expression level of mi R-10b-5p.However,overexpression of mi R-10b-5p in PBMCs reduced the expression of Th2 transcription factor GATA3 and inflammatory factors IL-4/IL-5/IL-13,and reduced the proportion of Th2 cells in CD4+T cells.The results of dual luciferase assay showed that the effect of mi R-10b-5p on lnc RNA and GATA3 was achieved by direct binding,respectively.The results of reversion experiment showed that overexpression of mi R-10b-5p in PBMCs with lnc-CEBPB-13overexpression resulted in decreased expression levels of GATA3,IL-4,IL-5 and IL-13compared with those with lnc-CEBPB-13 overexpression alone.These results indicated that mi R-10b-5p could alleviate the inflammatory response of Th2 induced by lnc-CEBPB-13.Conclusion:lnc-CEBPB-13 has a clear clinical relevance to AR patients,indicating that lnc-CEBPB-13 can be used as a biomarker for AR diagnosis in the future.lnc-CEBPB-13promoted Th2 cell differentiation through GATA3 expression.However,mi R-10b-5p plays a negative role in the regulation of Th2 cell differentiation and function by lnc-CEBPB-13.This effect is achieved by lnc-CEBPB-13 competing with mi R-10b-5 to regulate the expression of GATA3 and form ce RNA network.This study enriches the mechanism of the occurrence and development of AR and provides a scientific basis for the discovery of new therapeutic targets for AR.