Characterization of extracellular circulating microRNA

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 000g 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.

Figure 1.

(A) miRNAs but not detectable mRNA are present in conditioned media in nuclease resistant form. Conditioned media was collected from MCF7 cells and further treated as indicated on the graph. Cel-miR-39 (5 pg) was added into the media either before or after addition of denaturing Trizol LS reagent (Sigma). As a control for miRNA recovery, 5 pg of cel-miR-39 was dissolved in 30 μl of nuclease-free water. Total RNA from each sample was eluted in 30 μl of nuclease-free water and the content of miRNAs, β-actin and α-tubulin mRNAs were analyzed using quantitative real-time PCR as described in the ‘Materials and Methods’ section. Data presented as raw C_t values, each bar represents mean (SD) (n = 3). (B) Extracellular (EC) and intracellular (IC) levels of three miRNA species in five different cancer cell lines were measured by quantitative real-time PCR. Data presented as C_t values normalized on spiked in cel-miR-39 control; each bar represents mean (SD) of three independent RNA isolations. (n = 3). (C and D). Intra- and extra-cellular miRNA spectra for MCF-7 cells were measured by TaqMan miRNA Low density array (Applied Biosystems). (C). Correlation plot shows C_t values of miRNAs obtained from total RNA extracted from 400 μl of conditioned media and corresponding MCF7 cells monolayer. Those miRNAs that were not detected on the array (C_t > 35) were assigned C_t values of 40. EC—miRNAs presented exclusively in the cells; EM—miRNAs presented exclusively in the media (lists of exclusively expressed miRNAs Cp value <35 in Supplementary Table S1). (D) Those miRNAs that were detected in both cells and the media demonstrated a dramatic difference in extracellular/intracellular amount. Note, miRNAs content in the media was much lower [average ΔC_t (cell-media) = −7.5].

Figure 2.

(A) The procedure for the fractionation of human blood plasma and conditioned media. Cell-debris free (1200g following 14 000g centrifugation) blood plasma diluted 12 times in PBS and MCF7 cells conditioned media were used as starting samples. Microvesicles were removed from the samples by filtration through 0.22 μm filters; exosome-free fractions were obtained by ultracentrifugation at 110 000g. Furthermore, fractionation of 0.22 μm filtered samples was performed using 300, 100 and 50 kDa MWCO nanomembranes. (B) The efficacy of ultracentrifugation to remove exosomes and large proteins from plasma was assessed by SDS–PAGE gel of the 110 000g pellets. Coomassie blue (left) staining and western immunoblot (right) had shown that the first ultracentrifugation (PI pellet) removed large proteins (>300 kDa) and exosomes (as judged by exosomal marker protein CD9) from the plasma supernatant. While the pellet obtained after second ultracentrifugation of the same supernatant (PII) at the same force did not contain any detectable amounts of proteins including exosomal marker. (C and D) The level of three miRNA species miR-16, miR-21 and miR-24 was quantitatively assayed in the conditioned media (C) and blood plasma (D) samples after each fractionation step with individual TaqMan miRNA Assays. Spiked in cel-miR-39 was used as normalization control for all samples. Data presented as relative miRNA fold change. Each bar represents mean (SD) of three independent experiments (n = 3).

Figure 3.

miRNA spectra of the supernatants and the pellets obtained after ultracentrifugation of MCF7 cells conditioned media (A) and human blood plasma (B) as measured by TaqMan miRNA Low Density arrays (Applied Biosystems). Blood plasma (1 ml) was combined with 11 ml of 1×PBS before ultracentrifugation. ‘Left’: Correlation plots shows C_t values of miRNA signals obtained from total RNA extracted from 400 μl (1/30 volume) of supernatants and total pellets (pellets were obtained after ultracentrifugation of total 12 ml of samples). Note that only 1/30 of total supernatants were taken for the RNA isolation and subsequently for the TaqMan Low Density Arrays. MiRNAs which were undetected on the array (C_t > 35) were assigned C_t values of 40. ES—miRNAs presented exclusively in the supernatant; EP—miRNAs presented exclusively in the pellets (exclusively detected miRNAs are listed in Supplementary Data). ‘Right’: Those miRNAs that were detected in both supernatants and pellets demonstrated a dramatic difference in the content. Note: amount of miRNAs in the pellets is much lower than in supernatants.

Figure 4.

(A–C) The levels of extracellular miRNAs correlate with the level of extracellular Ago2 but not NPM1 protein. One-day serum-free conditioned media from MCF7 cells was fractionated using nanomembrane concentrators of 300, 100 and 50 kDa MWCO. (A) Individual TaqMan miRNA assays were used to quantify the species of miR-16, miR-21 and miR-24 which remained in the solution after each ultrafiltration step. (B) Concentrated fractions (retinates) were subjected to western immunoblot analysis using anti-Ago2 and anti-NPM1 antibody. Note: 300R = retinate from 300 kDa MWCO filter; 100R = 100 kDa retinate from 100 kDa MWCO filter and 50R = retinate from 50 kDa MWCO filter. (C) The amount of Ago2/miRNA in conditioned media was proportional to the amount of Ago2/miRNA in the cells. MCF7 cells lysate and concentrated serum-free conditioned media were prepared and miRNA content was assessed using TaqMan miRNA assays. After that, cell lysate was diluted so that the miRNA content in it equaled approximately the miRNA content in the concentrated serum-free media. Finally, western immunoblot was performed on concentrated media and diluted cell lysates and the amounts of Ago2 were visually compared. (D and E) Extracellular miRNA co-immunoprecipitated with anti-Ago2 antibody in a detergent-free environment. Species of miR-16, miR-21, miR-24 and exogenously expressed miR-34b were measured both in anti-Ago2 co-immunoprecipitates (P and P+Ago2) and in the remained supernatants (S and S+Ago2) obtained from MCF7 conditioned media (D) and blood plasma (E). Data presented as a fold change compared to control supernatants (assigned value 1), each bar represents mean (SD) of three independent experiments. Cel-miR-39 spiked in during RNA isolation step was used as normalization control. Note, the anti-Ago2 antibody induced a depletion of the miRNAs from conditioned media and blood plasma. (F) The levels of miR-16, miR-21 and miR-24 were measured in the anti-FLAG pellets obtained from HEK293T cells conditioned medias 72 h after transfection with plasmids-encoding FLAG-Ago1, FLAG-Ago2, FLAG-Ago3, FLAG-Ago4 or empty vector (K).

Figure 5.

Stability of miRNA and Ago2. (A) MCF7 cells were lysed in a buffer containing no protease or phosphatase inhibitors and either frozen or left at room temperature for 2 months. (B) Serum-free conditioned media obtained after incubation with MCF7 cells monolayer for 24 h was either frozen or left at RT for 2 months. The species of miRNAs and Ago2 content were analyzed by TaqMan miRNA qRT–PCR assays and western immunoblot, respectively, in both lysates and conditioned medias. Data presented as a fold change at 2 month point versus 0 point (frozen samples), each bar represents mean (SD) of three independent RNA isolations (n = 3). Cel-miR-39 was used as normalization control.