John R. Chevillet

MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non-vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2-miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.

Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.

Cell-free circulating microRNAs (miRNAs) in the blood are good diagnostic biomarker candidates for various physiopathological conditions, including cancer, neurodegeneration, diabetes and other diseases. Since their discovery in 2008 as blood biomarkers, the field has expanded rapidly with a number of important findings. Despite the initial optimistic views of their potential for clinical application, there are currently no circulating miRNA-based diagnostics in use. In this article, we review the status of circulating miRNAs, examine different analytical approaches, and address some of the challenges and opportunities.