Downregulation of miRNA-451a Promotes the Differentiation of CD4+ T Cells towards Th2 Cells by Upregulating ETS1 in Childhood Asthma

Abstract

Children exposed to common aeroallergens may develop asthma that progresses into adulthood. Inflammation regulated by T helper 2 (Th2) cells, a specific subpopulation of CD4+ T lymphocytes, is involved in asthmatic injury. Herein, our microarray data indicated that microRNA-451a-5p (miRNA-451a) expression decreased by 4.6-fold and ETS proto-oncogene 1 (ETS1) increased by 2.2-fold in the peripheral blood lymphocytes isolated from asthmatic children (n = 4) as compared to control individuals (n = 4). The negative correlation between miRNA-451a and ETS1 was further validated in 40 CD4+ T cell samples (10 healthy vs. 30 asthmatic samples). In vitro, naïve CD4+ T cells isolated from control individuals were cultured under Th2 cell polarizing condition. miRNA-451a expression decreased while ETS1 increased in CD4+ T cells in the setting of Th2 cell polarization. Moreover, miRNA-451a knockdown enhanced Th2 cell polarization - cells positive for both GATA3 (GATA binding protein 3, a Th2-transcription factor) and CD4 increased, and the generation of Th2 cell cytokines, interleukin (IL)5 and IL13, increased. In contrast, miRNA-451a overexpression inhibited Th2 cell differentiation. Interestingly, dual-Luciferase assay proved ETS1 as a novel target of miRNA-451a. Moreover, enforced expression of ETS1 partially restored miRNA-451a-induced inhibition of IL5 and IL13, and increased the GATA3+CD4+ cell population. Collectively, our work demonstrates that downregulation of miRNA-451a upregulates ETS1 expression in CD4+ T cells, which may contribute to Th2 cell differentiation in pediatric asthma.

Keywords: Childhood asthma; ETS proto-oncogene 1; Th2 cell differentiation; Type 2 inflammation; miRNA-451a.

Fig. 1

Downregulation of miRNA-451a and upregulation of ETS1 observed in lymphocytes derived from children with asthma (CA). a, c Volcano blots showed downregulated miRNAs as blue dots, and upregulated miRNAs as red dots. b, d All differentially expressed miRNAs and mRNAs are shown in heatmaps. Each row represents an miRNA or an mRNA, and each column indicates a sample. e The predictive targets of miR-451a with expression levels negatively correlated with miR-451a based on microarray data are shown in the Venn diagram. f The expression correlation between miR-451a and ETS1 CD4+ T cell samples from 10 control and 30 asthmatic children was analyzed with the Spearman correlation test (p < 0.05).

Fig. 2

Downregulation of miRNA-451a and upregulation of ETS1 are observed during the differentiation of CD4+ T cells towards Th2 cells. a Representative images of flow cytometry analysis showing GATA3+CD4+ cells. b qRT-PCR analysis of IL5 and IL13 in naïve CD4+ T cells under Th2 polarizing conditions. c The level of miRNA-451a and ETS1 was analyzed by qRT-PCR or Western blot. Statistically significant differences in b and c were analyzed by two-tailed unpaired t test (n = 3). ** p < 0.01, *** p < 0.001.

Fig. 3

miRNA-451a suppresses the differentiation of CD4+ T cells towards Th2 cells. a, c Representative images of flow cytometry analysis showing GATA3+CD4+ cells. b, d The protein levels of IL5 and IL13 in CD4+ T cells detected by Western blot. e, f ELISA analysis showed the level of IL5 and IL13 after silencing or overexpression of miRNA-451a. Data were analyzed by two-tailed unpaired t test or one-way ANOVA followed by Tukey's multiple comparation (n = 3). ** p < 0.01, *** p < 0.001. ns indicates a p value >0.05.

Fig. 4

miRNA-451a directly targets ETS1 in CD4+ T cells in vitro. a, c Miranda (http://www.microrna.org/microrna/home.do) and RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid) were used for target prediction. Sequence information of ETS1 WT and mutant-type (MT) 3′UTR. These fragments were inserted into pmirGLO plasmids, and co-transfected into 293T cells together with miRNA-451a mimics or NC mimics. b, d The relative luciferase activity was determined and analyzed with one-way ANOVA. Additionally, CD4+ T cells were induced with Th2 cell differentiation followed by infection with LV-anti-miRNA-451a and LV-miRNA-451a. The expression levels of ETS1 were analyzed by Western blot (e) and real-time qPCR (f). Data in e and f are shown as the mean ± SD from three separate experiments, with two-tailed unpaired t test (n = 3). ** p < 0.01, *** p < 0.001. NC, negative control.

Fig. 5

miRNA-451a inhibits Th2 cell differentiation through downregulating ETS1. Cells overexpressing miRNA-451a were infected with LV-OE-ETS1 or LV-OE-NC. a Representative images of flow cytometry analysis showing GATA3+CD4+ cells. b The mRNA levels of IL5 and IL13 were analyzed by qRT-PCR. c The protein expression of ETS1, IL5, and IL13 were determined with Western blot. d ELISA analysis of IL5 and IL13. The quantitative analyses in b and c are presented as the mean ± SD calculated based on three separate experiments with two-tailed unpaired t tests (n = 3). ** p < 0.01, *** p < 0.001. NC, negative control; OE, overexpression.