Fig. 2

Prediction and validation of ING5 as a direct target of miR-24. a Schematic description of the hypothetical duplex formed by the interactions between the binding site in the ING5 3’-UTR (top) and miR-24 (bottom). The seed region of miR-24 and the seed-recognition site in the ING5 3’-UTR are indicated in red. All nucleotides in the seed-recognition site are completely conserved in several species. The predicted free energy value of the hybrid is calculated. b qRT-PCR analysis of the expression levels of miR-24 in the same 8 pairs of BC and NC tissue samples. c-f qRT-PCR and western blot analysis in MCF-7 and MDA-MB-231 cells transfected with equal doses of scrambled negative control mimic (pre-miR-control), miR-24 mimic (pre-miR-24), scrambled negative control inhibitor (anti-miR-control) or miR-24 inhibitor (anti-miR-24). c: qRT-PCR analysis of miR-24; d: representative image of western blot analysis of ING5 protein; e: quantitative analysis of western blot; f: qRT-PCR analysis of ING5 mRNA. g-h Western blot analysis of the expression levels of ING5 protein in MCF-7 cells co-transfected with pre-miR-control plus control plasmid, pre-miR-24 plus control plasmid, pre-miR-control plus an ING5 overexpressing plasmid, or pre-miR-24 plus an ING5 overexpressing plasmid. g: representative image; h: quantitative analysis. i The relative luciferase activities in MCF-7 cells transfected with wild-type or mutant ING5 luciferase plasmid and with equal doses of pre-miR-control, pre-miR-24, anti-miR-control or anti-miR-24. *p < 0.05; **p < 0.01; ***p < 0.001