Export of microRNAs and microRNA-protective protein by mammalian cells

Abstract

The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research activities, but has left many unanswered questions about how this regulation functions and how it is integrated with other regulatory mechanisms. A number of miRNAs have been found to be present in plasma and other body fluids of humans and mice in surprisingly high concentrations. This observation was unexpected in two respects: first, the fact that these molecules are present at all outside the cell at significant concentrations and second, that these molecules appear to be stable outside of the cell. In light of this it has been suggested that the biological function of miRNAs may also extend outside of the cell and mediate cell-cell communication. We report here that after serum deprivation several human cell lines tested promptly export a substantial amount of miRNAs into the culture medium and the export process is largely energy dependent. The exported miRNAs are found both within and outside of the 16.5 and 120 K centrifugation pellets which contain most of the known cell-derived vesicles, the microvesicles and exosomes. We have identified some candidate proteins involved in this system, and one of these proteins may also play a role in protecting extracellular miRNAs from degradation. Our results point to a hitherto unrecognized and uncharacterized miRNA trafficking system in mammalian cells that is consistent with the cell-cell communication hypothesis.

Figure 1.

Differential miRNA spectra between cultured cells and the medium. (a) Intra- and extracellular miRNA spectra for six different cell lines in culture as measured by miRNA arrays. The cell lines are as indicated (T98 is derived from human glioblastoma multiforma, BEAS2B is derived from lung bronchial epithelium and HPF are primary pulmonary fibroblast cells isolated from human lung tissue). Heat maps represent the levels of miRNA for the cell lines as indicated (red highest, green lowest). The brackets indicating ‘in’ or ‘out’ mark groups of miRNAs that show striking differences between the intra (‘in’) and extra (‘out’) cellular concentrations specifically for HepG2 cells. (b) qPCR measurements of selected miRNAs shown in 1a that illustrate different profiles for HepG2 (red) and A549 (blue). The histogram shows the external versus internal levels of selected miRNAs. The extracellular levels are indicated above, the intracellular below.

Figure 2.

Time course of levels of miRNA exported into the medium. Samples were taken at various indicated time points after serum deprivation. The serum free medium contains no proteins or miRNAs. Most of the miRNA measured shown very similar behavior. The levels of 16 miRNAs measured by quantitative PCR from A549 (left frame) and HepG2 (right frame). The points are the means of three biological replicates. All vertical axes, the levels of miRNAs, are in ΔCt.

Figure 3.

Decay of miRNAs in the medium. The cells in culture were serum deprived for 2 h, then the medium was removed and incubated under the same conditions for varying periods of time. Specific miRNA levels were measured (three biological replicates) with quantitative PCR on each sample. (a) The time course measurements of four selected miRNAs with standard derivations showing diverse profiles. (b) Model kinetics for the export response of the majority of the miRNAs considered here, showing the rate of export as a function of time (in red) and the accumulated level of extracellular miRNA as a function of time (in blue). (c) the intracellular response in HepG2 cells of three selected miRNAs.

Figure 4.

miRNA levels in fractionated serum-free culture medium for A549 and HepG2. The medium, 2 h after serum deprivation, was fractionated by differential centrifugation into four fractions, microvesicles (size range: 100–1000 nm), exosomes (size range: 30–100 nm), one higher g spin pellet (220K for 1 h), and the remaining supernatant. The final supernant were concentrated to a final volume of 0.5 ml and 200 µl of the concentrated media were used for miRNA analysis. Pellets were also resuspended in 0.5 ml of PBS and 200 µl of the solution were used for miRNA isolation. (a) Experimental design for fractionation of medium. The levels of miRNA distribution from A549 (b) and HepG2 (c) were grouped into distinct distribution profiles.

Figure 4.

miRNA levels in fractionated serum-free culture medium for A549 and HepG2. The medium, 2 h after serum deprivation, was fractionated by differential centrifugation into four fractions, microvesicles (size range: 100–1000 nm), exosomes (size range: 30–100 nm), one higher g spin pellet (220K for 1 h), and the remaining supernatant. The final supernant were concentrated to a final volume of 0.5 ml and 200 µl of the concentrated media were used for miRNA analysis. Pellets were also resuspended in 0.5 ml of PBS and 200 µl of the solution were used for miRNA isolation. (a) Experimental design for fractionation of medium. The levels of miRNA distribution from A549 (b) and HepG2 (c) were grouped into distinct distribution profiles.

Figure 4.

miRNA levels in fractionated serum-free culture medium for A549 and HepG2. The medium, 2 h after serum deprivation, was fractionated by differential centrifugation into four fractions, microvesicles (size range: 100–1000 nm), exosomes (size range: 30–100 nm), one higher g spin pellet (220K for 1 h), and the remaining supernatant. The final supernant were concentrated to a final volume of 0.5 ml and 200 µl of the concentrated media were used for miRNA analysis. Pellets were also resuspended in 0.5 ml of PBS and 200 µl of the solution were used for miRNA isolation. (a) Experimental design for fractionation of medium. The levels of miRNA distribution from A549 (b) and HepG2 (c) were grouped into distinct distribution profiles.

Figure 5.

Protein exported upon serum deprivation. (a) NPM1 protein is observed in western analysis of the concentrated medium from HepG2 cells. This protein is exported into the medium, but is not found in any fraction except the supernatant (as defined in legend to Figure 4) as shown by westerns on the fractions. (b) The relative concentrations of all miRNAs measured in this work were averaged for each fraction to obtain a relative miRNA levels in each fraction. These averages for HepG2 cells are shown in the histogram using a scale of log to base 2. (c) NPM1 protein protects miRNA from RNase degradation. Synthetic mir-122 RNA (100 pmol) was mixed with different proteins, NPM1 (black bars, 3 pmol), TGF-β (gray bars, 4 pmol) or BSA (open bars, 1.5 nmol) for 30 min followed by adding RNase A (7 nmol) for another 30 min of incubation at 37°C. The miRNA levels were determined by qPCR. Control experiments: omitting RNase A, protein, or miRNA were also included, as indicated on top of the graph. The scale is Ct of relative mir-122 levels.