Figure 6. Inhibition of miR-362-5p acts through CYLD to suppress human NK cell function.

(a–b) Quantitative RT-PCR assay of miR-362-5p (a), and PRF1, GzmB, and IFNG (b) in sort-purified pNK cells transfected with synthetic FAM-labeled-miR-362-5p inhibitor (anti-miR-362-5p) or a synthetic control miRNA (Control). (c–d) Flow cytometry of the expression of perforin, granzyme-B, and IFN-γ; (c), and CD107a (d) in purified human pNK cells transfected with FAM-labeled-anti-miR-362-5p or negative control miRNA (Control). FAM positive pNK cells were gated and analyzed. The graphs show the average relative frequency of perforin⁺, granzyme-B⁺, IFN-γ, or CD107a⁺ pNK cells as determined above. (e) Flow cytometry assay evaluating the cytotoxic activity of pNK cells transfected with anti-miR-362-5p or control miRNA. Results are expressed as mean ± SEM of triplicate wells from one representative experiment of three experiments completed. (f–g) Flow cytometry analysis of the expression of perforin, granzyme-B and IFN-γ (f); and CD107a (g) in purified pNK cells transfected with FAM-anti-miR-362-5p in the presence or absence of CYLD siRNA. Representative FACS plots from FAM⁺ cells are shown. The graphs show the average relative frequency of all perforin⁺, granzyme-B⁺, IFN-γ⁺, or CD107a⁺ pNK cells as above. Data are representative of three independent experiments (mean ± SEM). *P < 0.05, **P < 0.01 and ***P < 0.005 (Student's t-test).