Characterization of extracellular circulating microRNA

TLDR

MicroRNAs have been detected in human body fluids as extracellular, nuclease‑resistant entities, yet their origin and function remain largely unknown. The authors hypothesize that extracellular miRNAs are mainly by‑products of dead cells that persist in the extracellular space because of the stability of the Ago2–miRNA complex. They found that circulating miRNAs are highly stable, non‑vesicular, and associated with Ago2 protein, with most miRNAs co‑immunoprecipitating with Ago2, indicating that extracellular miRNAs are predominantly exosome‑free, though some may associate with exosomes.

Abstract

MicroRNAs (miRNAs), a class of post-transcriptional gene expression regulators, have recently been detected in human body fluids, including peripheral blood plasma as extracellular nuclease resistant entities. However, the origin and function of extracellular circulating miRNA remain essentially unknown. Here, we confirmed that circulating mature miRNA in contrast to mRNA or snRNA is strikingly stable in blood plasma and cell culture media. Furthermore, we found that most miRNA in plasma and cell culture media completely passed through 0.22 µm filters but remained in the supernatant after ultracentrifugation at 110 000 g indicating the non-vesicular origin of the extracellular miRNA. Furthermore, western blot immunoassay revealed that extracellular miRNA ultrafiltrated together with the 96 kDa Ago2 protein, a part of RNA-induced silencing complex. Moreover, miRNAs in both blood plasma and cell culture media co-immunoprecipited with anti-Ago2 antibody in a detergent free environment. This is the first study to show that extracellular miRNAs are predominantly exosomes/microvesicles free and are associated with Ago proteins. We hypothesize that extracellular miRNAs are in the most part by-products of dead cells that remain in extracellular space due to the high stability of the Ago2 protein and Ago2-miRNA complex. Nevertheless, our data does not reject the possibility that some miRNAs can be associated with exosomes.