Fig. 4.

Exosomal transfer of MiR-126 from endothelial cells inhibits proliferation of malignant nasal-septum carcinoma (MNSC) cells. a Temporal and dose-dependent kinetics of HUVEC exosome uptake in MNSC cells, and (b) dose-response curve at 360 min. Exosomes (exo) were isolated from HUVECs grown in the medium supplemented with exosome-free serum. Isolated exosomes were stained with the lipophilic dye PKH-26 and extensively washed with two subsequent ultracentrifugation spins. Next, MNSC cells were incubated over time with PKH-26-labelled exosomes at different concentrations (μg/ml). Cell were then washed, trypsinized and their fluorescence analysed by flow cytometry, and the uptake was expressed as mean fluorescence intensity (MFI). c Representative fluorescence image of uptake of exosomes derived from HUVECs by MNSC cells. MNSC cells, cultured in exosome-depleted serum, were incubated with PKH-26 -labelled exosomes (20 μg/ml) from HUVECs for 4 h and their internalization visualized by fluorescent microscope (Zeiss, Axiocam MRc5; magnification 40-60×). The scale bar for all images equals 100 μm. d MiR-126 levels and cell proliferation of MNSC cells evaluated by the MTT assay after incubated with exosomes (20 μg/ml) isolated from HUVECs in the presence or absence of antiMiR. e Expression of MiR-126 targets IRS1, VEGF, SOX2, and EGFL7 after incubation of MNSC cells with exosomes isolated from HUVECs in the presence or absence of antiMiR. The data shown are mean values ± S.D. derived from three independent experiments. The symbol “*” denotes significant differences between non treated cells (CTRL) and exo-HUVEC treated cells with and without antiMiR, p < 0.05