High-throughput miRNA screening (total of 1728 miRNAs) was performed using the AccuTarget Human miRNA mimic library constructed on the basis of miRBase version 21 (Cosmo Bio, Tokyo, Japan). A 100-μl PC3M cell suspension of 5000 cells per well (in RPMI containing 10% serum without antibiotics) was seeded into 96 wells and incubated for 24 hours. Then, transfections of 10 nM miRNA mimics or siRNAs were accomplished with the DharmaFECT Transfection Reagent 1 (Dharmacon) according to the manufacturer’s protocol. After 24 hours, the medium was changed to advanced RPMI 1640 medium containing 2 mM l-glutamine without an antibiotic-antimycotic. Next, 48 hours after medium change, ExoScreen and cell growth assays, which are described above, were performed. Briefly, after 10 μl of CM was collected for the ExoScreen assay, cellular viability was examined through a cell growth assay. The values of EV secretion/cell viability were calculated using the results of the ExoScreen and cell growth assays. Then, the relative values of each well were calculated by comparing to the negative control well.

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