Allergy Asthma Immunol Res.  2015 Sep;7(5):449-457. 10.4168/aair.2015.7.5.449.

Altered microRNA Expression Profiles of Extracellular Vesicles in Nasal Mucus From Patients With Allergic Rhinitis

• Wu G
• Yang G
• Zhang R
• Xu G
• Zhang L
• Wen W
• Lu J
• Liu J
• Yu Y

• KMID: 2147953
• DOI: http://doi.org/10.4168/aair.2015.7.5.449

Abstract

PURPOSE
Allergic rhinitis (AR) is an inflammatory disorder of the upper airway. Exosomes or extracellular vesicles are nanosized vesicles of endosomal origin released from inflammatory and epithelial cells that have been implicated in allergic diseases. In this study, we characterized the microRNA (miRNA) content of exosomes in AR.
METHODS
Extracellular vesicles were isolated from nasal mucus from healthy control subjects (n=10) and patients with severe AR (n=10). Vesicle RNA was analyzed by using a TaqMan microRNA assays Human Panel-Early Access kit (Applied Biosystems, Foster City, CA, USA) containing probes for 366 human miRNAs, and selected findings were validated with quantitative RT-PCR. Target prediction and pathway analysis for the differentially expressed miRNAs were performed using DIANA-mirPath.
RESULTS
Twenty-one vesicle miRNAs were up-regulated and 14 miRNAs were under-regulated significantly (P<0.05) in nasal mucus from AR patients when compared to healthy controls. Bioinformatic analysis by DIANA-mirPath demonstrated that 32 KEGG biological processes were significantly enriched (P<0.05, FDR corrected) among differentially expressed vesicle miRNA signatures. Among them, the B-cell receptor signaling pathway (P=3.709E-09), the natural killer cell-mediated cytotoxicity (P=8.466E-05), the T-cell receptor signaling pathway (P=0.00075), the RIG-I-like receptor signaling pathway (P=0.00127), the Wnt signaling pathway (P=0.00130), endocytosis (P=0.00440), and salivary secretion (P=0.04660) were the most prominent pathways enriched in quantiles with differential vesicle miRNA patterns. Furthermore, miR-30-5p, miR-199b-3p, miR-874, miR-28-3p, miR-203, and miR-875-5p, involved in B-cell receptor and salivary secretion signaling pathways, were selected for validation using independent samples from 44 AR patients and 20 healthy controls. MiR-30-5p and miR-199b-3p were significantly increased in extracellular vesicles from nasal mucus when compared to healthy controls, while miR-874 and miR-28-3p were significantly down-regulated. In addition, miRNA-203 was significantly increased in AR patients, while miRNA-875-5p was found to be significantly decreased in AR patients.
CONCLUSIONS
This study demonstrated that vesicle miRNA may be a regulator for the development of AR.

Keyword

Extracellular vesicles; microRNAs; allergic rhinitis

MeSH Terms

B-Lymphocytes
Biological Processes
Endocytosis
Epithelial Cells
Exosomes
Humans
MicroRNAs*
Mucus*
Receptors, Antigen, T-Cell
Rhinitis*
RNA
Wnt Signaling Pathway
MicroRNAs
RNA
Receptors, Antigen, T-Cell

Figure

• 

  Fig. 1 Flow cytometric analysis of extracellular vesicles displayed expression of CD63 and MHCII surface markers. Results are shown as the MFI for the detected molecule divided by the MFI for the isotype control. Data shown are representative of at least 3 independent experiments and are shown as mean±SEM.

• 

  Fig. 2 Correlation between TaqMan microRNA and TaqMan real-time RT-PCR assays. Eight detectable miRNAs (miR-146a, miR-155, let-7a, miR-181a, miR-454, let-7b, miR-28-3p, and miR-885-5p) were selected for the evaluation of the 384-well microfluidic card, by independent TaqMan real-time RT-PCR on the same samples. Data are represented representative of 3 repetitive experiments.

• 

  Fig. 3 Differentially expressed vesicle miRNAs in the B-cell receptor signaling pathway. (A) Diagram of the B-cell receptor signal pathway. Four dysregulated miRNAs (miR-30-5p, miR-199b-3p, miR-874, and miR-28-3p) were associated with the B-cell receptor signaling pathway by possibly regulating their potential gene targets. (B) Quantitative expression of miR-30-5p, miR-199b-3p, miR-874, and miR-28-3p as assessed by TaqMan real-time RT-PCR in individual nasal mucus vesicle samples from 44 AR patients and 20 healthy controls. Relative expression of these miRNAs was statistically differentially expressed compared to healthy controls (P<0.05, Mann Whitney U test). All qRT-PCR reactions were performed in triplicate, and the Ct values greater than 35 from real-time PCR assays were treated as 35. The delta Ct values were calculated by using RNU44 as the endogenous control. *P<0.05; **P<0.01.

• 

  Fig. 4 Differentially expressed vesicle miRNAs in the salivary secretion signal pathway. (A) Two dysregulated miRNAs (miR-203 and miR-875-5p) as examples for examining miRNA-mRNA relationships in salivary secretion. (B) Quantitative expression of miR-203 and miR-875-5p as assessed by TaqMan real-time RT-PCR in individual nasal mucus vesicle samples from 44 AR patients and 20 healthy controls. Relative expression of both miRNAs was statistically differentially expressed compared to healthy controls (P<0.05, Mann Whitney U test). All qRT-PCR reactions were performed in triplicate, and the Ct values greater than 35 from the real-time PCR assays were treated as 35. The delta Ct values were calculated by using RNU44 as the endogenous control. *P<0.05; **P<0.01.

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