MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression

Abstract

Allergic airway inflammation is characterized by marked in situ changes in gene and protein expression, yet the role of microRNAs (miRNAs), a new family of key mRNA regulatory molecules, in this process has not yet been reported. Using a highly sensitive microarray-based approach, we identified 21 miRNAs with differential expression between doxycycline-induced lung-specific IL-13 transgenic mice (with allergic airway inflammation) and control mice. In particular, we observed overexpression of miR-21 and underexpression of miR-1 in the induced IL-13 transgenic mice compared with control mice. These findings were validated in two independent models of allergen-induced allergic airway inflammation and in IL-4 lung transgenic mice. Although IL-13-induced miR-21 expression was IL-13Ralpha1 dependent, allergen-induced miR-21 expression was mediated mainly independent of IL-13Ralpha1 and STAT6. Notably, predictive algorithms identified potential direct miR-21 targets among IL-13-regulated lung transcripts, such as IL-12p35 mRNA, which was decreased in IL-13 transgenic mice. Introduction of pre-miR-21 dose dependently inhibited cellular expression of a reporter vector harboring the 3'-untranslated region of IL-12p35. Moreover, mutating miR-21 binding sites in IL-12p35 3'-untranslated region abrogated miR-21-mediated repression. In summary, we have identified a miRNA signature in allergic airway inflammation, which includes miR-21 that modulates IL-12, a molecule germane to Th cell polarization.

Figures

Figure 1. MiRNA expression profile in IL-13 transgenic mice lung

(A) Heat map of 21 differentially expressed miRNAs following control no doxycycline (−) and doxycycline (+) exposure for 28 days. Relative expression is log2 transformed. (B) A qRT-PCR validation of a selected set of miRNA probes normalized to snoRNA202. NS, not significant; *, p < 0.05; ***, p < 0.001, ****, p < 0.0001. (C) Correlation of miRNA microarray and qRT-PCR validation; dashed line represents 95% confidence interval. Data are represented as mean ± S.E.M; n = 5–7 mice per group; data representative of 3 experiments.

Figure 2. Expression of miR-21 and miR-1 in experimental asthma models

MiR-21 (A) and miR-1 (B) expression were assessed in OVA and Aspergillus fumigatus asthma models. (C) MiR-21 expression was determined in IL-4 lung transgenic mice. The relative expression levels were determined by qRT-PCR normalized to snoRNA202; *, p < 0.05; **, p < 0.01, ***, p < 0.001; ****, p < 0.0001. Data are represented as mean ± S.E.M; n = 3–7 mice per group; data representative of 3 experiments.

Figure 3. Expression of miR-21 in allergen challenged IL-13 receptor alpha 1 deficient mice

(A) MiR-21 expression was determined in IL-13 challenged wild type and IL-13 receptor alpha 1 deficient mice. (B–C) MiR-21 expression was determined in OVA (B) and Aspergillus fumigatus (Asp) models (C) using IL-13 receptor alpha 1 (−/−) mice and wild type (+/+) controls. (D) MiR-21 expression was determined in OVA challenged wild type and STAT6 deficient mice. The relative expression levels were determined by qRT-PCR normalized to snoRNA202. NS, not significant; **, p < 0.01, ***, p < 0.001. Data are represented as mean ± S.E.M.; n = 3–8 mice per group; data representative of 3 experiments.

Figure 4. In situ hybridization of miR-21 in Aspergillus fumigatus challenged wild type mouse lung

Expression of miR-21 in (A–B) Aspergillus fumigatus challenged wild type mouse lungs, were determined by LNA-based in situ hybridization. (A) LNA-anti-miR-21, left: 100X field; right: 400× field; (B) Left: 200× field, LNA-anti-miR-21. Right: serial sections at 600×; right top: LNA-anti-miR-21, right middle: anti-CD68, right bottom: LNA-scrambled control probe. (C) Relative expression of miR-21 in different cell types; BM DC: bone marrow derived dendritic cells; BM Mac: bone marrow derived macrophages; BALF: bronchoalveolar lavage fluid cells; MLE15: murine lung epithelial cell line; MFLM4: murine lung mensenchyme cell line; NIH 3T3: murine fibroblasts. (D) Relative expression of miR-21 in LPS stimulated murine macrophage cell line Raw264.7; ***, p < 0.001. Data are representative of three experiments.

Figure 5. MiR-21 targets IL-12p35

(A) Predicted highly conserved binding site for miR-21 in 3’UTR of IL-12p35. The 8-mer seed sequence is shaded in gray. (B) IL-12p35 expression was determined in OVA, Aspergillus fumigatus (Asp), and doxcycline induced IL-13 bitransgenic models. The relative expression levels were determined by qRT-PCR normalized to HPRT1. *, p < 0.05; **, p < 0.01; n = 5–7 mice per group. (C) Relative luciferase activity in 293T cells co-transfected with control firefly luciferase vector (pMIR-Report), or a firefly luciferase reporter vector containing the 3’UTR of IL-12p35 (pmIL12p35), or a firefly luciferase vector with perfect miR-21 binding site in the 3’UTR (pMIR-21), and either the pre-miR-21 expression vector (pMIRNA1-Pre-miR-21) or control vector (pMIRNA1-Control). Firefly luciferase activity was normalized to the renilla luciferase activity then to the average of the control firefly luciferase reporter; NS, not significant; ***, p < 0.001; n =4 per group, data representative of 3 experiments. (D) A dose response study of pre-miR-21 expression vector on the luciferase activity of the luciferase vector containing the 3’UTR of IL-12p35; n = 4 per group, data representative of 3 experiments. (E) Mutation (yellow-highlighting) in the 3’UTR of mIL-12p35. (F) Relative luciferase activity in 293T cells co-transfected with reporter plasmid containing either the wild type or mutant mIL-12p35 3’UTR. Firefly luciferase activity was normalized to the renilla luciferase activity then to the average of the wild type mIL-12p35 firefly luciferase reporter; NS, not significant; ***, p < 0.001; n = 4 per group, data representative of 3 experiments. All data are represented as mean ± S.E.M.