Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma

Abstract

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.icroRNAs (miRNAs) are a class of approximately 22 nucleotide noncoding RNAs that mediate posttranscriptional gene regulation by binding to and repressing specific messenger RNA targets. We and others previously demonstrated that miRNAs are present in the human circulation in a cell-free form and that altered plasma and serum miRNA profiles are observed in cancer and other diseases (1-9). This, along with the finding that miRNAs are remarkably stable in plasma despite high circulating RNase activity (1), suggests that miRNAs may be developed into a powerful new class of blood-based biomarkers.The mechanism underlying the unexpected stability of cell-free miRNAs in the RNase-rich environment of blood has not been systematically investigated, although it has important implications for miRNA biomarker development and for potential biological functions of circulating miRNAs (10). Currently, the dominant model for circulating miRNA stability is that miRNAs are released from cells in membrane-bound vesicles, which protect them from blood RNase activity. Vesicles proposed as carriers of circulating miRNAs include exosomes, which are 50-to 90-nm vesicles arising from multivesicular bodie...

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

“…To this end, the abundance of the EV-associated miRNA let-7a and of the Argonaute 2-associated miR-16, reported as being excluded from EVs [45,55], was analysed.…”

Blood-Based Biomarkers for Metabolic Syndrome

“…Conversely, an analysis of three miRNAs in plasma found them to be predominantly (>97%) exosome-free [24]. Furthermore, Arroyo et al [25] claimed that 90% of miRNAs in the circulation are not present in a membrane-bound form. However, regardless of whether or not miRNAs are contained within EVs, it is apparent that blood-based miRNAs have promise as biomarkers for full-blown MetS and for many of its contributing components (see section on Circulating miRNA Biomarkers for MetS).…”

Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

“…A separate first step isolation method is SEC systems, which allow for little more than 20 mL input. Finally, use of crowding reagents, particularly on fluids such as serum/plasma, tend to co-precipitate large proteins and ribonucleic acid:protein complexes such as Ago-complexed miRNA [28]. These contaminants can lead to EV circulating DNA and long RNAs also precipitate under these conditions, making it difficult to assign a biological phenotype to any one molecular entity.…”