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.

Fig. 1.

Circulating miRNAs are not restricted to vesicles. (A) Circulating vesicles were purified from plasma by differential ultracentrifugation. Recovery of vesicles consistent in size and morphology with exosomes and microvesicles was confirmed by EM. A representative micrograph is shown. (B) The plasma vesicle pellet (gray bars) and vesicle-poor supernatant (white bars) from the indicated donors were assayed for miR-16, miR-92a, and let-7a by using TaqMan qRT-PCR. For each miRNA, a standard curve of the corresponding synthetic oligonucleotide enabled absolute quantification of miRNA copies. Bars represent relative copies of miRNA recovered from the pellet and supernatant.

Fig. 2.

Two populations of circulating miRNAs are identified by size-exclusion chromatography. (A–C) Plasma samples from the indicated donors were fractionated on a Sephacryl S-500 column. Fractions were assayed for miR-16 (blue diamond), miR-92a (orange triangle), and let-7a (red square) by using absolute quantification by TaqMan qRT-PCR. Protein abundance was determined by absorbance at 280 nm (A280; black circle). For miRNAs, the y-axis values indicate relative copies of miRNA in each fraction. For protein, the same y-axis values indicate A280 reading. (D) Elution profiles of vesicles (100 nm mean diameter), BSA, and tyrosine standards were determined by A280. Points represent the mean ± SD of duplicate independent analyses.

Fig. 3.

A protease-sensitive complex protects late-eluting miRNAs from plasma RNase activity. Plasma was untreated (closed symbols) or treated with proteinase K (5 mg/mL; open symbols) at 55 °C. At times indicated, aliquots were removed and assayed for miR-16 (A), miR-92a (B), and let-7a (C) by qRT-PCR. Points represent miRNA copies detected at each time relative to the corresponding 0-min sample.

Fig. 4.

Circulating miRNAs are predominantly in fractions consistent with ribonucleoprotein complexes. (A) RQ of circulating miRNAs profiled in the indicated plasma fraction pools (donor H1-7) is presented as a heat map. Shading represents miRNA level detected in a given fraction pool relative to the total of that miRNA detected across all pools. Above the heat map is a composite of the vesicle standard and corresponding plasma protein elution profile from Fig. 2 (vesicle and protein A280 values are scaled so peaks are the same height). Hierarchical clustering ordered miRNAs as indicated by the dendrogram. Assay names are colored by three classes: early-eluting (red), late-eluting (blue), and eluting in both peaks (purple). Boldface miRNAs were measured by individual TaqMan qRT-PCR assays. Validation of miR-142–3p (B), miR-150 (C), and miR-122 (D) levels in fractions of plasma (blue diamonds) and serum (red squares) from donor H1-7. Points represent the miRNA relative copies in fractions as determined by TaqMan qRT-PCR and absolute quantification.

Fig. 5.

Cell-free circulating Ago2–miRNA ribonucleoprotein complexes exist in human plasma. (A) Ago2 immunoprecipitated from fresh platelet-poor plasma from three healthy donors was detected by immunoblotting. Immunoprecipitation with normal mouse IgG served as a negative control. 293T cell lysate was a positive control for Ago2. (B) Immunoprecipitates in A were assayed for the indicated miRNAs. Bars represent the proportion of miRNA that was recovered in the immunoprecipitate from the plasma volume used for immunoprecipitation. Bars labeled “All Donors” represent the mean ± SD of the three donors.

Fig. 6.

Late-eluting miRNAs are specifically present within circulating Ago2 ribonucleoprotein complexes. (A) Relative copies of miR-16, miR-92a, and let-7a in each plasma fraction shown in Fig. 2 A–C were averaged (Upper). Points represent the mean ± SD of the three donors. Ago2 immunoprecipitated from the same plasma fractions of the indicated donors was detected by immunoblotting (Lower). 293T cell lysate (lane marked “+”) was a positive control for immunoblotting. Fraction numbers refer to both panels. (B) Plasma fraction Ago2 immunoprecipitates in A were assayed for the indicated miRNAs. The y axes correspond to the proportion of each miRNA recovered in the Ago2 immunoprecipitate of a given fraction relative to the total of that miRNA detected across all plasma fractions for the respective donor. Points represent the mean ± SD from three donors. (C) For each donor, the cumulative miRNA detected in Ago2 immunoprecipitates of all plasma fractions relative to the total miRNA detected in all plasma fractions was calculated. Bars represent the mean ± SD for three donors.