miR-21 is induced by IL-6 via Stat3 activation and promotes survival of myeloma cells. (A) XG-1 and INA-6 cells were either restimulated with IL-6 for the times indicated after cytokine withdrawal for 72 and 12 hours, respectively, or continuously cultured with IL-6 (perm.). Levels of pri-miR-21 were determined by real-time PCR. Values obtained for cells deprived of IL-6 were set to 1. (B) XG-1 and INA-6 cells cultured in the presence of IL-6 were transiently transfected with an expression plasmid for a small hairpin RNA-silencing Stat3 (▩) or a scrambled sequence RNA (□) together with a vector-encoding enhanced GFP. Successful down-regulation of Stat3 protein levels under these conditions has been demonstrated by us previously.⁹  After 48 hours, green fluorescent cells were sorted, and Stat3 and pri-miR-21 transcript levels were determined by real-time PCR. Values for the control samples were taken as reference. (C) In XG-1 cells treated as were INA-6 cells, mature miR-21 was quantified by stem-loop reverse transcription followed by real-time PCR.¹⁰  (D,E) INA-6 cells were transiently transfected by electroporation with a control (D) or a miR-21 expression vector (E). An expression plasmid encoding enhanced GFP was cotransfected. At 1 day after transfection, cell culture was continued either in the presence or absence of IL-6. After an additional 24 or 48 hours, apoptosis was studied by flow cytometric annexin-V assay, with the transfected cells gated on the basis of green fluorescence. In panel D, the reduced levels of viable (annexin-V⁻) cells after cytokine withdrawal are represented relative to those observed in IL-6–treated cells. Panel E shows the relative percentage of viable cells with miR-21 expression vector relative to control vector transfection. Data represent mean values plus or minus SD from 3 independent experiments.