Purification and Characterization of MicroRNAs Within Middle Ear Fluid Exosomes: Implication in Otitis Media Pathophysiology

Overview

Journal Pediatr Res

Specialties Biology
Pediatrics

Date 2017 Feb 4

PMID 28157838

Citations 11

Authors

Stephanie Val

Stephanie Jeong

Marian Poley

Anna Krueger

Gustavo Nino

Kristy Brown

Diego Preciado

Affiliations

Soon will be listed here.

Abstract

Background: Otitis media (OM) is characterized by acute infection progressing to chronic middle ear effusion (MEE). Extracellular secretion of microRNAs (miRNAs) in exosomes is a newly discovered mechanism for cells exerting distant cell genetic regulation. Whether MEE contains exosomes with specific miRNAs is unknown. This study aimed to purify and characterize the exosomal and miRNA content of MEE.

Method: MEEs were subjected to Exoquick exosomal purification and EXOCET exosomal quantification. Extracted vesicles were analyzed by dynamic light scattering (DLS), transmission electron microscopy (TEM), and immunoblotting of HSP-70. NanoString hybridization was performed to profile miRNAs. Exosomal protein content was profiled by Liquid chromatography tandem mass spectrometry (LC-MS/MS).

Results: EXOCET assays showed presence of exosomes (0-0.5 × 10/ml) in MEEs. DLS confirmed exosomal size between 10 and 200 nm. Western blot analysis showed presence of HSP-70. Twenty-nine miRNAs were found to be unique to MEEs. The most abundant miRNA was miR-223, a miRNA typically secreted by neutrophils. Proteomics demonstrated typical neutrophil markers as well as common innate immune molecules.

Conclusion: To our knowledge, this the first report demonstrating the presence of exosomes transporting miRNAs in MEEs. These findings open a broad and novel area of research in OM pathophysiology as driven by miRNA cell communication.

Citing Articles

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Proteomic Study of Middle Ear Effusion and Its Clinical Application for Otitis Media with Effusion.

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