Fig. 5: miR-483-3p suppresses a BCLAF1/PUMA/BAK1 signaling network.

A RT-qPCR of miR-483-3p and miR-483-5p following MIR483 overexpression in DU145 cells. B Dual luciferase assay of wild-type (WT) and seed-mutant (MUT) PUMA 3’UTR (left and middle), and WT BAK1 3’UTR reporters in DU145 cells. Schematic representation of the miR-483-3p binding site in the PUMA 3’UTR (top right) and the miR-483-3p seed mutation (bottom right). C Schematic representation of ENCODE ChIPseq peaks and the promoter regions of BBC3 (top) and BAK1 (bottom) from GM12878 cells (red track; GSE105550) and K562 cells (blue track; GSE105733). Alignment of ChIPseq peaks and promoter regions is highlighted in red. D Western blot of BCLAF1 in MIR483 DKO DU145-Cas9 cells. Representative blot is shown on the left and quantitation is shown on the right. E Same as (D) in PC3-Cas9 cells. F Same as (B) with WT, 6-mer MUT, 8-mer MUT and double MUT BCLAF1 3’UTR reporter. Schematic representation of the miR-483-3p binding sites in the BCLAF1 3’UTR (top right) and the miR-483-3p seed mutations (bottom). G Western blot of BCLAF1, PUMA, and BAK1 in MIR483 overexpressing DU145 cells. Representative blot is shown on the left and quantitation is shown on the right. All data represented as mean ± SEM from n = 3 independent experiments. P values obtained using an Unpaired one-tailed Student’s t test (A, D, E, G) or a two-way ANOVA with a Holm-Sidak’s multiple comparisons test (B, F). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.