Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology.
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.


METHODS
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 × 107/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.


CONCLUSIONS
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.
Sheikh Zayed Center for Pediatric Surgical Innovation, Children's National Health System, Washington, DC.
Stéphanie
S
Val
Sheikh Zayed Center for Pediatric Surgical Innovation, Children's National Health System, Washington, DC.
Stephanie
S
Jeong
Sheikh Zayed Center for Pediatric Surgical Innovation, Children's National Health System, Washington, DC.
Marian
M
Poley
Sheikh Zayed Center for Pediatric Surgical Innovation, Children's National Health System, Washington, DC.
Anna
A
Krueger
Center for Genetic Medicine Research, Children's National Health System, Washington, DC.|Division of Pediatric Pulmonology, Children's National Health System, Washington, DC.
Gustavo
G
Nino
Center for Genetic Medicine Research, Children's National Health System, Washington, DC.
Kristy
K
Brown
Sheikh Zayed Center for Pediatric Surgical Innovation, Children's National Health System, Washington, DC.|Division of Pediatric Otolaryngology, Children's National Health System, Washington, DC.
Diego
D
Preciado
D015153:Blotting, Western; D002853:Chromatography, Liquid; D055354:Exosomes / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D013058:Mass Spectrometry; D035683:MicroRNAs / Q000302:isolation & purification* / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D010033:Otitis Media / Q000378:metabolism* / Q000503:physiopathology; D016133:Polymerase Chain Reaction; D040901:Proteomics
D035683:MicroRNAs