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

Abstract

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.

Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle‐ear epithelial cells

“…Importantly, some of the miRNAs present in HMEEC secretions were also reported by us to be found in MEEs from patients with COM (18). These are listed in Supplemental Table 4.…”

“…Somewhat surprisingly, the typically intracellular heterogeneous ribonucleoproteins A2B1 (hnRNP A2B1) and K (hnRNP K) were enriched in secretions at 24 h (fold inductions of 1.99 and 1.78) and hnRNP Q at 48 h (fold induction 4.73). Given our recent publication identifying miRNA‐laden exosomes within MEE samples (18) and the fact that hnRNPs are known to function in the packaging of exosomes (31), we elected to validate further this finding of the hnRNP presence in HMEEC in vitro secretions.…”

“…They are endocytic vesicles transporting miRNAs, proteins, and mRNAs and function as putative mediators of intercellular communication. We recently were the first group to characterize their presence within human middle-ear fluid samples from patients with COM (18). In this study, we used multiple techniques to characterize the presence of exosomes in in vitro HMEEC secretions, allowing for the comparison of these with what we reported in the in vivo patient samples.…”

“…Methods previously reported for the isolation of exosomes from MEEs (18) were used to isolate exosomes from HMEEC secretions as follows. The whole secretions of HMEECs (40 ml per condition) were centrifuged at 10,000 g for 10 min at 4°C to remove debris, filtered with a 0.22‐µm membrane to remove bigger vesicles, and concentrated with an Amicon‐3K column (Millipore) to concentrate proteins >3 kDa.…”

“…They are thought to play an important role in cell-cell communication and immune response. We recently reported the presence of miRNAs in MEEs from patients with COM, with a high abundance of miRNA (miR)-223 related to the presence of neutrophils in the middle ear (18). This specific miRNA is known to be part of the signature of activated neutrophils (19), but the cellular origin of the other miRNAs detected in middle-ear fluid could not be determined.…”