Human miR-27a* suppresses both Prf1 and GzmB expression. (A) Venn diagram of the predictions of human Prf1- and GzmB-targeting miRNAs yielding one common hit miR-27a*. (B) Validation of microarray data with real-time qPCR and kinetic analysis of candidate miRNAs during NK-cell activation (0, 6, 12, and 24 hours). (C) Screening of functional miRNA(s) in suppression of Prf1 and GzmB protein expressions. One hundred microliters of 1 nucleofection sample contained 4 × 106 mNK cells and 100nM (final concentration) of miRNA mimics and was subjected to nucleofection. (Top) IB analysis. (Bottom) Standard Cr release assay at ET5. (D) Regulation of Prf1 and GzmB expression by miR-27a*. mNK cells were transfected with increasing concentrations (50 and 100nM) of miR-27a* or miR-27a* inhibitor. The transfected cells were incubated in the presence of IL-15 (30 ng/mL) for 24 hours and then further incubated with freshly added IL-15 (30 ng/mL) for 24 hours. Finally, cells were subjected to IB analysis and real-time qPCR. (Top) IB analysis. (Bottom) Real-time qPCR analysis. Data are representative of 3 independent experiments (mean ± SEM of triplicates; ns, not significant; Student t test, P > .05 and ***P < .001 vs Ctrl_miR).
