Fig. 2

The negative regulation of Survivin expression by miR-138-5p in bladder cancer cells. a Schematic description of the hypothetical hybridizations formed by the interactions between the binding sites in the BIRC5 3′-UTR (top) and miR-138-5p (bottom). The seed regions of miR-138-5p and the seed-recognition sites in the BIRC5 3′-UTR are indicated in red. All the nucleotides in the seed-recognition sites are completely conserved in several species. The predicted free energy values of each hybrid are indicated. b Quantitative RT-PCR analysis of the relative expression levels (miR-138-5p vs. U6) of miR-138-5p in human bladder cancer specimens (BC) and normal adjacent tissues (NAT). c Quantitative RT-PCR analysis of the expression levels of miR-138-5p in T24 and J82 cells transfected with equal doses of the miR-138-5p mimic (mim-miR-138-5p), miR-138-5p inhibitor (anti-miR-138-5p) or corresponding scrambled negative control RNA (mim-scramble and anti-scramble, respectively). d and e Western blotting analysis to detect Survivin protein levels in T24 and J82 cells transfected with equal amounts of mim-miR-138-5p, anti-miR-138-5p, mim-scramble or anti-scramble. d: representative image; e: quantitative analysis. f Quantitative RT-PCR analysis of Survivin mRNA levels in T24 and J82 cells transfected with equal amounts of mim-miR-138-5p, anti-miR-138-5p, mim-scramble or anti-scramble. g Direct recognition of the Survivin 3’-UTR by miR-138-5p. Firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-138-5p binding sites in the Survivin 3′-UTR were co-transfected with equal doses of mim-miR-138-5p, anti-miR-138-5p or the corresponding scrambled negative control RNA (mim-scramble and anti-scramble, respectively) into HEK293 cells. At 24 hours after transfection, the cells were assayed using a luciferase assay kit. Firefly luciferase values were normalized to β-galactosidase activity, and the results were calculated as the ratio of firefly luciferase activity in the miR-138-5p-transfected cells normalized to the activity in the negative control RNA-transfected cells. The results are presented as the mean ± S.D. of three independent experiments. *p < 0.05; ** p < 0.01; *** p < 0.005