MiR-365-3p is a negative regulator in IL-17-mediated asthmatic inflammation

Abstract

Background: Interleukin-17, the major proinflammatory cytokine secreted by Th17 cells, makes essential contribution to pathogenesis of severe asthma, while the detailed mechanisms, especially the involvement of microRNAs which are also important participants in asthma progression, remains largely unclear.

Methods: In this study, we established a house dust mite (HDM) extract-induced murine asthmatic models and the miRNA expression in the lung tissues of mice were profiled by miRNA microarray assay. The effect of miR-365-3p on IL-17-mediated inflammation was examined by qRT-PCR and immunoblotting analysis. The involvement of ARRB2 as target gene of miR-365-3p was verified by overexpression or RNA interference.

Results: HDM extract-induced asthmatic inflammation was proved to be IL17-mediated and miR-365-3p was screened out to be the only miRNA exclusively responsive to IL-17. miR-365-3p, whose expression was significantly downregulated upon IL-17 stimulation, was demonstrated to exert remarkable anti-inflammatory effect to decrease IL-17-provoked inflammatory cytokines (KC/IL-8 and IL-6) in both airway epithelial cells and macrophages of murine and human origins, verifying its universal antagonizing activity against IL-17-initiated inflammation across the two species. ARRB2 was characterized as the key target of miR-365-3p to negate IL-17-induced inflammatory cytokines.

Conclusion: Taken together, our data supported the notion that miR-365-3p, which was diminished by IL-17 in murine and human asthmatic pathogenesis, functioned as an essential negative mediator in IL-17-stimuated inflammatory response by targeting ARRB2, which would shed new light to the understanding and therapeutics thereof of asthmatic inflammation.

Keywords: ARRB2; IL-17KO; IL-8; asthma; miR-365-3p.

Figure 1

IL-17 is necessary to maintain asthmatic phenotype in HDM-induced murine model. (A) The level of IL-17A in mouse Bronchoalveolar lavage fluid (BALF) detected by Bio-Plex. N=10. (B, C) IHC staining (B) and quantitative analysis (C) of IL-17 positive cells in mouse lung tissue section. The brown area represents IL-17 positive. Black arrows indicate stained macrophage. Scale bar =50μm. N=10. (D, E) IHC staining (D) and quantitative analysis (E) of airway smooth muscle mass (indicated by Alpha-Smooth Muscle Actin) in mouse lung tissue section. The brown areas represent the airway smooth muscle cells. Scale bars= 100μm. N=10. (F, G) collagen deposition analysis in mouse lung tissue. Collagen was stained by Picro Sirius red, the color red indicates the positive staining. Scale bars=100μm. N=10. *, P<0.05, **, P<0.01, ***, P<0.001. n.s., not statistically significant.

Figure 2

Indispensable role of IL-17 in the inflammatory response of HDM-induced asthma in murine model. (A) Counts of inflammatory cells in BALF. N=10. (B) The level of inflammatory cytokines in BALF. The inflammatory cytokines in BALF were detected by Bio-Plex. N=10. *, P<0.05, **,P<0.01, ***, P<0.001. n.s., not statistically significant.

Figure 3

miR-365-3p is downregulated in wildtype HDM-induced mice but not in IL-17 KO HDM-induced mice. (A) Microarray of dysregulated miRNA in wildtype saline and HDM-induced mice, N=3. (B) The level of miR-365-3p examined by qRT-PCR in wild-type and IL-17KO mice. N=10. (C) The effect of IL-17 in miR-365-3p expression in MLE-12 cells. The MLE-12 cells were treated by IL-17, and the miR-365-3p expression was tested by qRT-PCR N=4. *, P<0.05, **, P<0.01, ***, P<0.001. n.s., not statistically significant.

Figure 4

miR-365-3p negates IL-17-induced inflammatory cytokines expression in murine epithelial cells and macrophages. (A) Effect of miR-365-3p mimic on mRNA expression of KC and IL-6 in MLE-12 cells upon IL-17 treatment. N=5. (B) Effect of miR-365-3p mimic on mRNA expression of IL-6 in J774a.1 cells upon IL-17 treatment. N=3. *, P<0.05, ***, P<0.001. n.s., not statistically significant.

Figure 5

miR-365-3p also serves as a negative regulator in hIL-17 induced inflammatory response in human airway epithelial cells and macrophages. (A) Sequence comparison of mmu-miR-365-3p and has-miR-365a-3p. (B) Effect of miR-365-3p mimic on mRNA expression of IL-8 and IL-6 in human A549 cells upon hIL-17 treatment. N=3. (C) Effect of miR-365-3p mimic on mRNA expression of IL-6 in PMA-differentiated U937 cells upon hIL-17 treatment. N=3. *, P<0.05, **,P<0.01, ***, P<0.001. n.s., not statistically significant.

Figure 6

ARRB2 is the target gene of miR-365-3p to negate proinflammatory effect of IL-17. (A) target genes of miR-365-3p that are related with asthma. (B) mRNA expression of ARRB2 detected by qRT-PCR. N=5. (C) The targeting role of miR-365-3p on ARRB2 examined by reporter fluorescence assays, N=3. (D) Effect of miR-365-3p rescue on mRNA level of ARRB2. N=6. (E) Effect of miR-365-3p rescue on protein level of ARRB2. (F) regulatory effect of miR-365-3p on ARRB2 expression in both mouse MLE-12 (left) and human A549 (right) epithelial cells. (G) immunoblotting analysis of ARRB2 overexpression from pcDNA3.1 vector in MLE-12 cells. (H) expression of KC and IL-6 in ARRB2-overexpression MLE-12 cells upon IL-17 treatment. N=4. (I) immunoblotting analysis of ARRB2 knockdown by siRNA in MLE-12 cells. (J) expression of KC and IL-6 in ARRB2-knockdown MLE-12 cells upon IL-17 treatment. N=4. *, P<0.05, **, P<0.01, ***, P<0.001. n.s., not statistically significant.

Figure 7

The proposed scheme of negative regulating role of miR-365-3p in IL-17 induced KC and IL-6 production. miR-365-3p, which was diminished by IL-17 in murine and human asthmatic pathogenesis, functioned as an essential negative mediator to suppress IL8 (KC for mouse) and IL-6 upon IL-17 stimulation by targeting ARRB2.