Cellular uptake of functional miR-223 contained in microvesicles from GM-CSF–stimulated monocytes does not require recipient cell transcriptional activity for expression. (A) A549, CCL-204, or HUVEC cells were incubated with microvesicles from GM-CSF–stimulated monocytes for 2 days. RNA was then isolated and expression for miRs-223, -29b, and -34a was measured by qRT-PCR. Shown is the average fold increase ± SEM (n = 4) in miR-223 levels compared with untreated cells. A significant increase after treatment with GM-CSF–stimulated microvesicles is observed for miR-223 (*P < .05), while miRs-29b and -34a levels remained unchanged. (B) A549 cells (5 × 106 cells/condition) were treated with vehicle (DMSO) or actinomycin D at the indicated concentrations for 6 hours. The cells were washed and culture media was replaced in the absence or presence of microvesicles. After 24 hours, RNA was isolated and miR-223 expression measured by qRT-PCR. Fold increase was determined by comparing the samples to the cells treated only with DMSO. Shown is the average ± SEM for cells treated with microvesicles from 4 independent donors. (C) A549 cells were transfected with the luciferase reporter vector containing miR-223 recognition sequence, a control luciferase vector lacking the sequence or cotransfected with miR-223 precursor. After 18-20 hours incubation, the cells were treated with or without microvesicles from GM-CSF–stimulated monocytes. The cells were cultured for another 24 hours then lysed and the luciferase activity was measured. The data are expressed as fold-decrease of luciferase activity of cells transfected with the luciferase reporter containing miR-223 recognition sequence over the vector lacking the recognition sequence for each culture condition. Shown is the average data ± SEM from A549 cells treated with microvesicles generated from 4 independent monocyte-derived microvesicle donors.
