miR-132, miR-212, and miR-200a negatively regulate STAT4 expression and IL-12–induced IFN-γ production in NK cells. (A) Human NK cells (2 × 105) were transfected with control mimics (miR ctrl), miR-491-5p, miR-132, miR-212, miR-200a, or an equimolar mixture of miR-132, miR-212, and miR-200a (miR mix) mimics as indicated at an aggregate final concentration of 50nM. After 48 hours culture in the presence of IL-2, the expression of STAT4 was measured by flow cytometry. Data are representative of 3 independent experiments with NK cells from different donors. (B) Summary of data from panel A, mean of STAT4 levels in indicated miRNA overexpression cells were analyzed to determine the relative effects of different miRNA on STAT4 protein levels. The results are STAT4 MFI mean ± SEM of representative experiment with NK cells from 3 different donors. *P < .05 versus miR ctrl. (C) Cells treated as in panel A (but not harvested) were washed with complete growth medium and cultured with or without 10 ng/mL of IL-12 for another 12 hours in the presence of IL-2. Cellular expression of phosphorylated STAT4 was measured by flow cytometry. Data are representative of 3 independent experiments with NK cells from different donors. (D) Summary of data from panel C. Mean of p-STAT4 levels in indicated miRNA overexpression cells was analyzed to determine the relative effects of different miRNA on IL-12–induced STAT4 phosphorylation. The results are p-STAT4 MFI mean ± SEM of representative experiment with NK cells from 3 different donors. *P < .05 versus miR ctrl. (E) Cells treated as in panel C. Cellular expression of IFN-γ was measured by flow cytometry. Data are representative of 3 independent experiments with NK cells from different donors. (F) Summary of data from panel E. Mean of IL-12–induced IFN-γ expression in indicated miRNA overexpression cells (IFN-γ MFI with IL-12 – IFN-γ MFI without IL-12) was calculated to determine the relative effects of different miRNA on IL-12–induced IFN-γ expression. The results are increased IFN-γ MFI mean ± SEM of representative experiment with NK cells from 3 different donors. *P < .05, **P < .01 versus miR ctrl. The gray dotted histogram represents the isotype control staining of NK cells.
