Human bone marrow-mesenchymal stem cell-derived exosomal microRNA-188 reduces bronchial smooth muscle cell proliferation in asthma through suppressing the JARID2/Wnt/β-catenin axis

Abstract

The functions of exosomes in allergic diseases including asthma have aroused increasing concerns. This paper focuses on the effects of exosomes derived from human bone marrow-mesenchymal stem cells (hBM-MSCs) on the proliferation of bronchial smooth muscle cells in asthma and the mechanism involved. Exosomes were extracted from hBM-MSCs and identified. Human BSMCs were induced with transforming growth factor (TGF)-β1 to mimic an asthma-like condition in vitro and then treated with exosomes. A mouse model with asthma was induced by ovalbumin (OVA) and treated with exosomes for in vivo study. The hBM-MSC-derived exosomes significantly reduced the abnormal proliferation and migration of TGF-β1-treated BSMCs. microRNA (miR)-188 was the most enriched miRNA in exosomes according the microarray analysis, and JARID2 was identified as a mRNA target of miR-188. Either downregulation of miR-188 or upregulation of JARID2 blocked the protective effects of exosomes on BSMCs. JARID2 activated the Wnt/β-catenin signaling pathway. In the asthmatic mice, hBM-MSC-derived exosomes reduced inflammatory cell infiltration, mucus production, and collagen deposition in mouse lung tissues. In conclusion, this study suggestes that hBM-MSC-derived exosomes suppress proliferation of BSMCs and lung injury in asthmatic mice through the miR-188/JARID2/Wnt/β-catenin axis. This study may provide novel insights into asthma management.

Keywords: Human bone marrow-mesenchymal stem cells; JARID2; asthma; bronchial smooth muscle cells; exosomes; microRNA-188; wnt/β-catenin.

Figure 1.

Identification of the hBM-MSCs and the derived exosomes. (a), expression of the surface marker proteins of hBM-MSCs determined by flow cytometry; (b), osteogenic and adipogenic differentiation abilities of hBM-MSCs examined by alizarin red staining (left) and oil red O staining (right), respectively; (c), shapes of the extracted particles observed under a TEM; (d), distribution of the particle size determined by nanoparticle tracking analysis; (e), protein levels of exosome-specific marker proteins CD81 and TSG101 determined by Western blot analysis. Three independent experiments were performed.

Figure 2.

Exosomes inhibit proliferation and migration of TGF-β1-induced BSMCs. (a), absorption of exosomes by BMSCs examined by PKH26 labeling (one-way ANOVA, *p < 0.05 vs 0 h); (b), proliferation of cells determined by the CCK-8 method (two-way ANOVA, #p < 0.05 vs control; &p < 0.05 vs model); (c), DNA replication activity of cells determined by EdU labeling assay (one-way ANOVA, #p < 0.05 vs control; &p < 0.05 vs model); (d), apoptosis of cells determined by flow cytometry (one-way ANOVA, #p < 0.05 vs control; &p < 0.05 vs model); (e), migration ability of cells measured by Transwell assay (one-way ANOVA, #p < 0.05 vs control; &p < 0.05 vs model). Data were presented as mean ± SD from three independent experiments.

Figure 3.

Exosomes convey miR-188 into BSMCs. (a), impact of different concentration of exosomes on the hybridization effect of miRNA microarrays (one-way ANOVA, *p < 0.05 vs 5 μg); (b), top five differentially expressed miRNAs in BSMCs after exosome treatment; (c), miR-188 expression in exosome resuspension and in the supernatant collected after the final centrifugation determined by RT-qPCR (unpaired t test, ###p < 0.001 vs supernatant); (d), in-situ miR-188 expression in BSMCs after exosome treatment determined by the FISH assay (one-way ANOVA, @p < 0.05 vs 0 h); (e), relative abundance of the five miRNAs in exosomes examined by RT-qPCR (one-way ANOVA, &p < 0.05 vs miR-188); (f), basic expression of the five miRNAs in TGF-β1-treated BMSCs (&p < 0.05 vs miR-188). Data were presented as mean ± SD from three independent experiments.

Figure 4.

Downregulation of miR-188 blocks the effects of exosomes on BSMCs. (a), expression of miR-188 in hBM-MSCs after miR-188 inhibitor transfection determined by RT-qPCR (unpaired t test, *p < 0.05); (b), miR-188 expression in exo-inhibitor and in exo-NC determined by RT-qPCR (unpaired t test, #p < 0.05); (c), in-situ miR-188 expression in BSMCs after exosome treatment determined by the FISH assay (unpaired t test, #p < 0.05); (d), proliferation of cells determined by the CCK-8 method (unpaired t test, #p < 0.05); (e), DNA replication activity of cells determined by EdU labeling assay (unpaired t test, #p < 0.05); (f), apoptosis of cells determined by flow cytometry (unpaired t test, #p < 0.05); (g), migration ability of cells measured by Transwell assay (unpaired t test, #p < 0.05). Data were presented as mean ± SD from three independent experiments.

Figure 5.

miR-188 directly targets JARID2. (a), a Venn diagram for the intersected target mRNAs of miR-188 predicted using five bioinformatic systems; (b), transfection efficiency of miR-188 in BSMCs determined by RT-qPCR (unpaired t test, *p < 0.05); (c), mRNA expression of UBR7, JARID2, FOXN2 and CD2AP in cells detected by RT-qPCR (two-way ANOVA,*p < 0.05); (d), protein expression of JARID2 in cells measured by Western blot analysis (unpaired t test, *p < 0.05); (e), putative binding sequence between miR-188 and JARID2 obtained from StarBase; (f), binding relationship between miR-188 and JARID2 validated through a dual luciferase reporter gene assay (two-way ANOVA,*p < 0.05); (g), binding relationship between miR-188 and JARID2 validated through an RIP assay (two-way ANOVA, *p < 0.05, **p < 0.01). Data were presented as mean ± SD from three independent experiments.

Figure 6.

Overexpression of JARID2 activates the Wnt/β-catenin pathway and blocks the effects of exosomes on BSMCs. (a), mRNA expression of JARID2 in hBM-MSCs after pcDNA-JARID2 transfection and XAV-939 treatment determined by RT-qPCR (one-way ANOVA, *p < 0.05 vs exo + pcDNA); (b), protein level of β-catenin in cells evaluated by Western blot analysis (one-way ANOVA, #p < 0.05 vs control, @p < 0.05 vs model, *p < 0.05 vs exo + pcDNA, &p < 0.05 vs exo + pcDNA-JARID2 + DMSO); (c), proliferation of cells determined by the CCK-8 method (two-way ANOVA, *p < 0.05 vs exo + pcDNA; &p < 0.05 vs exo + pcDNA-JARID2 + DMSO); (d), DNA replication activity of cells determined by EdU labeling assay (one-way ANOVA, *p < 0.05 vs exo + pcDNA; &p < 0.05 vs exo + pcDNA-JARID2 + DMSO); (e), apoptosis of cells determined by flow cytometry (one-way ANOVA, *p < 0.05 vs exo + pcDNA group; &p < 0.05 vs exo + pcDNA-JARID2 + DMSO); (f), migration ability of cells measured by Transwell assay (one-way ANOVA, *p < 0.05 vs exo + pcDNA; &p < 0.05 vs exo + pcDNA-JARID2 + DMSO). Data were presented as mean ± SD from three independent experiments.

Figure 7.

Exosomes relieve lung injury in mice induced with OVA. (a), a diagram for the establishment of the mouse model with OVA-induced asthma and the time points for different treatments; (b), expression of miR-188 and mRNA expression of JARID2 and β-catenin in the homogenate of mouse lung tissues determined by RT-qPCR (two-way ANOVA, *p < 0.05 vs control, #p < 0.05 vs OVA); (c), expression of miR-188 in the lung airway tissues of mice examined by the FISH assay (one-way ANOVA, *p < 0.05 vs control, #p < 0.05 vs OVA); (d), pathological changes in mouse lung tissues determined by HE staining and PAS staining. N = 10 in each group. Data were presented as mean ± SD from three independent experiments.

Figure 8.

The study roadmap.