Inhibition of miR-24 increases c-MYC, induces apoptosis, and reduces cell proliferation in U2932 DLBCL. (A) Expression of c-MYC in U2932 MPA vector (left panel) and in U2932 EBNA2 (right panel). EBNA2 expression increases c-MYC in U2932, which is further increased upon inhibition of miR-24 (right panel). β-actin was used as a loading control. Densitometry of blots is shown in supplemental Figure 1C. (B) The percentage of apoptotic cells in U2932 MPA vector and EBNA2 cells transfected with control inhibitors and anti–miR-24 was performed by using PE annexin V staining with 7-amino-actinomycin (7-AAD) vital dye. The graphs represent annexin V and 7AAD density plots, showing a gradient increase of the cell distribution. Cells stained only with annexin V are in early apoptosis. (C) The histograms represent the percentage annexin V–positive cells from 3 independent experiments. Statistical analysis was performed using ordinary one-way ANOVA and Tukey multiple comparisons test. (D) Inhibition of miR-24 reduces cell proliferation. In total, 2 × 105 cells per mL were seeded and counted daily. Trypan blue exclusion assay was performed to estimate the number of cells per mL at 24, 48, 72, and 96 hours, after transfecting U2932 vector or EBNA2 transfectants with control inhibitor or anti–miR-24. Statistical significance was calculated with ordinary one-way ANOVA and Tukey multiple comparisons test. At 48 hours, ∗∗P < .01, and at 96 hours, ∗∗∗P < .001, ∗∗∗∗P < .0001. (E) Schematic representation of miR-24 increase by EBNA2 for rheostatic maintenance of proproliferative c-MYC (left) and therapeutic potential of anti–miR-24 molecules (right).
