MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells

Abstract

MicroRNAs provide an additional layer in the regulation of gene expression acting as repressors with several targets at the posttranscriptional level. This study describes microRNA expression patterns during differentiation and activation of mast cells. The expression levels of 567 different mouse miRNAs were compared by microarray between c-Kit+ committed progenitors, mucosal mast cells, resting and IgE-crosslinked BMMCs in vitro. The strongest upregulation of miR-132 upon IgE-mediated activation was validated in human cord blood-derived mast cells as well. HB-EGF growth factor also upregulated upon activation and was ranked high by more prediction algorithms. Co-transfection of miR-132 mimicking precursor and the 3'UTR of human Hbegf-containing luciferase vector proves that the predicted binding site is functional. In line with this, neutralization of miR-132 by anti-miR inhibitor leads to sustained production of HB-EGF protein in activated mast cells. Our data provide a novel example for negative regulation of a growth factor by an upregulated miRNA.

Fig. 1

The heat map of differentially expressed miRNA genes among murine mast cell samples representing mucosal mast cell differentiation and activation. a Data passing the signal intensity filter and showing at least two-fold significant changes between BMMCs and c-Kit+ progenitors were hierarchically clustered (entities and conditions, Euclidean metric centroid linkage). b Differentially expressed miRNAs upon IgE-crosslinking were clustered in a similar way. Blue color indicates the relative downregulation, red shows upregulation in the respective group

Fig. 2

Validation of microarray data by qRT-PCR. a–e qRT-PCR validation of a selected set of miRNA probes normalized to snoRNA135. *^, **^, ****^{, #} indicate p-values <0.05, <0.01, <0.001, <10⁻⁵, respectively. Data are represented as mean ± SEM; n = 4–6 samples per group. f Correlation of miRNA microarray and qRT-PCR data. Dashed line represents 95% confidence interval (Spearman rank order correlation R = 0.857, p < 0.05)

Fig. 3

miR-132 is upregulated upon IgE-crosslink in mouse and human mast cells. a Kinetics of Il13 and miR-132 upregulation after ionomycin treatment in MC/9 mouse mast cell line. The induction of miR-132 reaches its plateau phase 2 h after ionomycin treatment (N = 3). b miR-132 is upregulated in human cord blood-derived mast cells 2 h after IgE-crosslinking. (n = 4, p < 0.05, unpaired T test; mean ± SEM)

Fig. 4

Target prediction of miR-132 using mouse and human data sets of three different target prediction databases. The size of outputs from the predictions and their overlap for the human (a) and mouse (b) database show surprisingly minimal overlap. c Targets included in at least two outputs. d Genes from c were graded according to the sum of ranked scores. The color intensity indicates the relative probability of the interaction between miR-132 and the respective targets. Values in each box show the fractionated ranks in the given data set with HUGO identifiers on the right side

Fig. 5

MiR-132 has two predicted binding sites in the 3′UTR of mouse and human HB-EGF. a The target prediction database TargetScan (version 5.1) shows an 8mer and a 7mer-1A predicted pairing between human HB-EGF 3′UTR and miR-132. b The predicted target sites are identical in the mouse and human HB-EGF 3′UTRs. c Minimum free energy values and structures of the hybridized miRNA/target duplexes calculated by RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/)

Fig. 6

Kinetic analysis of HB-EGF production in activated mouse mast cells. IgE-presensitized BMMCs were stimulated by 10 ng/ml DNP-HSA, and samples were collected both for RNA and protein analysis from the same cultures at the indicated time points. Note that the marked upregulation of Hbegf mRNA (a, relative expression on a logarithmic scale) is followed by subsequent protein production (b), which returned to baseline after 24 h. The densitometry results of three biological parallels are shown (mean ± SEM). A representative Western blot is depicted on c. d Hbegf is upregulated in human cord blood-derived mast cells 2 h after IgE-crosslinking, as well (n = 5, p < 0.001, unpaired T test, mean ± SEM)

Fig. 7

miR-132 regulates HB-EGF expression at protein level. a Pre-miR-132 represses the activity of HB-EGF 3′UTR luciferase vector. CHO cells were co-transfected with a luciferase vector containing HB-EGF 3′UTR or control sequence and negative control precursor oligos or Pre-miR-132. The measured luciferase activity signals were normalized to values of the only with vector transfected cells, and then these values were plotted as ratios of correspondingly treated groups, dividing normalized signals derived from HB-EGF 3′UTR and control sequences containing luciferase (n = 3, means ± SEM are shown, **p < 0.01 by ANOVA and Tukey-HSD post-hoc). b Kinetics of miR-132 and Hbegf expression in MC/9 mast cells transfected with miR-132 mimicking (Pre-miR) and neutralizing (Anti-miR) oligonucleotides. MC/9 cells were activated by ionomycin for the indicated time intervals (n = 3, mean ± SEM). Note that miR-132 did not influence the expression of Hbegf at the RNA level in MC/9 mouse cell line. c The effect of modulated expression of miR-132 on HB-EGF protein level in BMMCs. Three independently differentiated BMMCs were pooled and transfected with 100 nM Anti-miR negative control (NC), Anti-miR-132 and Pre-miR-132 oligonucleotides with Amaxa Nucleofector. After 24 h, cells were presensitized with IgE, and then 10 ng/ml DNP-HSA was added. The cells were lysed after the indicated incubation time, and then equal amounts of total proteins were subjected to Western blotting. The blots were incubated with the same buffer with corresponding antibodies (anti-HB-EGF or anti-actin) and exposed to the same X-ray film in order to compare the inter-transfection baseline alterations. d Quantification of the bands on Fig. 7c. These bands were quantified with ImageJ, and the HBEGF/actin density values were plotted after normalization to the corresponding controls. The x axis shows the time point after IgE+ antigen activation. Note that the HB-EGF protein expression was still sustained in the Anti-miR-132-transfected samples even after 72-h-long stimulation