Fig. 5

E2F1 is a direct target of miR-183-5p|+2. a Schematic representation of the different psiCHECK-2 constructs in 3’UTR reporter luciferase assay and the human E2F1 mRNA indicating the binding site for miR-183-5p|+2. b Validation of direct targeting of E2F1 3’UTR by 3’UTR luciferase assay. MDA-MB-231 and BT-549 were seeded in white 96-well plates. psiCHECK-2, psiCHECK-2_E2F1_3’UTR or mutated psiCHECK-2_E2F1_3’UTR plasmid was cotransfected with miRNA mimics or miRNA negative control for 48 h. The cells were lysed and the activity of renilla and firefly luciferase was measured using GloMax Microplate Reader. Renilla luciferase measurements were normalized to the activity of firefly luciferase. The relative luciferase activity was normalized to the empty psiCHECK-2. Thereafter, values were normalized to miRNA negative control. c-d Downregulation of E2F1 by miR-183-5p|+2 overexpression at mRNA and protein level. MDA-MB-231 and BT-549 were seeded in 6-well plates and transfected with miRNA mimics (miR-183-5p|0, miR-183-5p|+1 and miR-183-5p|+2) or siAllstar (as miRNA negative control) for 48 h. c RNA was isolated using RNeasy kit (Qiagen). The mRNA expression levels of target genes were quantified by Taqman qRT-PCR. Gene expression was normalized to housekeeping genes (ACTB and GAPDH) using the ddCt method. Normalized gene expression is depicted as the relative expression compared to cells transfected with siAllstar. d Cells were lysed in RIPA buffer and protein expression level of E2F1 and GAPDH was determined by western blot. Representative blots are depicted. Band intensities were quantified using ImageStudio with median background subtraction method. The expression of E2F1 was normalized to GAPDH. The values are depicted as relative expression compared to cells transfected with siAllstar. Data are presented as mean ± SD, n = 3 (each derived from the median of 3 technical replicates). Statistical analysis: ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test; *p < 0.05, ** p < 0.01, ***p < 0.001 compared to control