Characterization of CLL exosome RNA and transfer of functional miRNAs to target cells. (A) RNA was extracted from CLL cells and exosomes and analyzed using the Agilent Bioanalyzer RNA (left and middle) and small RNA (right) chips. (B and C) Small RNA next-generation sequencing of RNA purified from CLL cells and exosomes (MEC-1). (B) Percentages of the various small RNA categories identified in CLL cells and exosomes. (C) Percentages of the 10 most abundant miRNAs in CLL-exosomes. (D and E) Primary BM-MSCs from healthy donors (HD) were treated with 50 µg/mL CLL exosomes (MEC-1) for the indicated times, and specific miRNAs were quantified by qRT-PCR. Data are presented as fold change (FC) relative to untreated cells (Ctrl). (D) Kinetic quantification of miR-150 (n = 3). (E) Quantification of miR-146a, miR-150, and miR-155 after 72 hours. (F) Quantification of miR-146a and miR-150 in BM-MSCs (2 × 10⁴) cocultured with primary CLL cells (1 × 10⁶ in upper compartment) in 0.4-µm pore inserts for 24 hours by qRT-PCR. Data are presented as FC relative to BM-MSCs alone (Ctrl). (G) qRT-PCR quantification of miR-150 in BM-MSCs incubated with DMEM (Ctrl), DMEM supplemented with 20% fetal calf serum, HD, or CLL plasma for 3 hours (n = 3). (H) HMEC-1 cells were transfected with luciferase reporter plasmids carrying miR-146a or miR-150 antisense sequences (pmiR146aAS or pmiR150AS) and then cultured in the absence (Ctrl) or presence of MEC-1 exosomes (Exo) for 24 hours. Luciferase activity of reporter plasmid was quantified by measuring the light emission (RLU) of both luciferases in 4 replicates per condition. Data are presented as RLU ratio relative to Ctrl (n = 3). *P < .05, ***P < .001.