miR-10a directly targets Bcl-6 and Ncor2 and down-regulates protein expression. (a) miR-10a target sequences (in red) of the 3'UTR of the Bcl6 (upper) and Ncor2 (lower) mRNAs, which were included in luciferase reporter vectors (left panels). Also shown are the mutant vectors, in which the miR-10a seed complementary sequences were deleted as indicated by asterisks (Luc-Bcl6-del and Luc-Ncor2-del). These luciferase reporter constructs were transfected with miR-10a or a control construct in NIH3T3 cells and luciferase expression was determined and normalized by Renilla luciferase activity. (b, c) CH12, a B cell lymphoma cell line that expresses Bcl-6, was transduced with retroviral vectors encoding hNGFR and miR-10a (blue) or a control vector (red). Bcl-6 expression was measured by flow cytometry after gating hNGFR+ population (b) or by immunoblotting after transduced (hNGFR+) cells were isolated by magnetic beads (c). Blotting of lysates with anti-actin antibody is shown below with quantification of Bcl-6/actin ratios determined by densitometry. (d – h) Naïve CD4+ T cells were transduced with control vectors (red) or vectors expressing miR-10a (blue) and stimulated with anti-CD3 and CD28 antibodies with TGF-β (5 ng/ml) and IL-2 (50 U/ml) and anti-IFN-γ antibody (iTreg condition). Bcl-6 levels were assessed by flow cytometry after gating on transduced cells (d). A representative result is shown on the left panel and levels of Bcl-6 expression expressed as the average mean fluorescence intensity are shown on the right. (e, h) The effect miR-10a on levels of Bcl-6 and Ncor2 in activated CD4+ T cells transduced with miR-10a and control vector was confirmed by immunoblotting and quantified as in (c). (f, i) Bcl-6 and Ncor2 mRNA expression was measured by quantitative RT-PCR and normalized to β-actin mRNA level. (g) NIH3T3 cells were transduced with different combinations of three vectors: a Flag-tagged Bcl-6 expression vector that includes the Bcl-6 3'UTR and hNGFR; a vector encoding miR-10a and Orange2; and a vector encoding the miR-10a sponge target sequence and Δ4GFP. Cells were also transduced with corresponding control vectors as delineated in the figure. Changes in Bcl-6 levels were measured by flow cytometry using anti-Flag antibody after gating on GFP+Orange2+hNGFR+ cells and are expressed as MFI. (j, k) Naïve CD4+ T cells were transduced with a control vector or a vector expressing a miR-10a sponge target sequence (miR-10a-5pT) and stimulated under iTreg-inducing conditions. Bcl-6 expression in CD4+hNGFR+ population was evaluated by flow cytometry and the MFI is shown in the right panel (j). Transduced cells were isolated and Ncor2 expression was assessed by immunoblotting. Pooled data from three independent experiments were shown in the right panel (k). Similar results were obtained from three (a, e – k) or two (b, c, d) individual experiments.