Small RNA Sequencing Reveals Exosomal miRNAs Involved in the Treatment of Asthma by Scorpio and Centipede

Abstract

Asthma is a common respiratory disease with inflammation in the lungs. Exosomes and microRNAs (miRNAs) play crucial role in inflammation, whereas the role of exosomal miRNA in asthma remains unknown. Here, we aimed to identify the key exosomal miRNAs and their underlying mechanisms involved in scorpio and centipede (SC) treatment in asthma. Eighteen mice were randomly divided into three groups: control group, asthma group, and SC treatment group. Effect of SC was assessed by hematoxylin-eosin staining and real-time PCR. Exosomes from asthma and SC treatment groups were analyzed by small RNA-seq. Results revealed SC significantly alleviated the pathogenesis of asthma and suppressed the release of inflammatory cytokines. A total of 328 exosomal miRNAs were differentially expressed between the exosomes from asthma and SC-treated mice, including 118 up- and 210 downregulated in SC-treated mice. The altered exosomal miRNAs were primarily involved in the function of transcription, apoptotic process, and cell adhesion; and pathway of calcium, Wnt, and MAPK signaling. Real-time PCR verified exosomal miR-147 was downregulated, while miR-98-5p and miR-10a-5p were upregulated in SC-treated mice compared to asthma mice. Moreover, the target genes of miR-147-3p, miR-98-5p, and miR-10a-5p were mainly enriched in Wnt and MAPK inflammatory signaling. miR-10a-5p promoted the proliferation of mouse lung epithelial cells and downregulated the expression of Nfat5 and Map2k6. These data suggest SC-induced exosomal miRNAs might mediate the inflammatory signaling and might be involved in the SC treatment in asthma. The exosomal miRNAs might be promising candidates for the treatment of asthma.

Figure 1

Scorpio and centipede (SC) improved pathology and inflammation of asthma in mice. (a–c) Histopathological analysis was performed by hematoxylin-eosin (H&E) staining. Enlargement factor is 200 (d and e). Expression of inflammatory factor was detected by real-time PCR. n = 6, two samples t-test, ^∗∗P < 0.01.

Figure 2

Exosomal identification and small RNA sequencing data. (a) Exosomes were analyzed by transmission electron microscopy (scale bar, 100 nm). (b) Western blot was used to measure the expression of exosomal marker in lung tissues and exosomes from bronchoalveolar lavage fluid of mice. (c) Length distribution of esosomal microRNAs (miRNAs). (d) Principal component analysis (PCA) of exosomal miRNAs from asthma group and SC treatment group. (e) Venn diagram analysis of common and unique exosomal miRNAs in the asthma group and SC treatment group. Model represents asthma group and SC represents SC treatment group.

Figure 3

Analysis of differentially expressed miRNAs in exosomes between asthma group and SC treatment group. (a) Volcano plot analysis of differentially expressed miRNAs in bronchoalveolar lavage fluid- (BALF-) derived exosomes between the asthma group and SC treatment group. The blue point represents a downregulated exosomal miRNA and the red point represents an upregulated exosomal miRNA in the SC treatment group compared to the asthma group. (b) Heat map analysis differentially expressed miRNAs in BALF-derived exosomes between asthma group and SC treatment group. Green represents low expression level, red represents high expression level.

Figure 4

Function and pathway analysis of target genes of different exosomal miRNAs. (a) Target gene analysis of differentially expressed miRNAs. Number represents the target gene numbers of differentially expressed miRNAs using RNAhybrid and Miranda. (b) Gene Ontology (GO) analysis for target genes of differentially expressed miRNAs in BALF-derived exosomes between asthma group and SC treatment group. (c) Kyoto Encyclopedia of Genes and Genomes (KEGG) for target genes of differentially expressed miRNAs in BALF-derived exosomes between the asthma group and SC treatment group. The circle size represents the number of enriched genes and the color represents P value.

Figure 5

Expression and target gene analysis of the key exosomal miRNAs. (a) Candidate exosomal miRNAs were verified by real time PCR. n = 6, two samples t-test, ^∗P < 0.05. (b) Change tendency comparison between the results of real-time PCR and small RNA-seq. Fold change: the fold change in SC treatment group compared to the asthma group. (c) Network analysis of miRNA-mRNA-pathway. Triangles, circles, and boxes represent miRNA, mRNA, and pathway, respectively. Red represents upregulated and green represents downregulated in SC treatment group compared to the asthma group.

Figure 6

Functional and target gene validation of mmu-miR-10a-5p. (a) The transfection effect of mmu-miR-10a-5p mimics and inhibitor in mouse lung epithelial cells was verified by real-time PCR. (b) The effect of mmu-miR-10a-5p on proliferation of mouse lung epithelial cells was verified by CCK-8 assays. (c) The target gene expression of mmu-miR-10a-5p was verified by real-time PCR. n = 3, two samples t-test, ^∗P < 0.05, ^∗∗P < 0.01.