Figure 4

IGF2BP1 is a direct target of miR-196b in HepG2 cells. (A) The putative miR-196b targeted sequence in the igf2bp1 gene. TargetScan6.2 predicts three binding sites in IGF2BP1 3′-UTR. The alignment of the seed region of miR-196b with 3′UTR is shown. (B) Expression level of IGF2BP1 mRNA in the pre-miR-196b transfected cells determined by RT-qPCR was down-regulated in comparison to pre-miR negative control transfected cells (pre-miR CTL-) or untransfected cells (Cells), 24 or 48 h post-transfection (means ± 1 SD, n = 3). °, °°: significantly different from untransfected cells (p < 0.05, p < 0.01), $$$: significantly different from pre-miR negative control transfected cells (p < 0.001), for each group (N, H, NE, HE) respectively. (C) Protein abundance of IGF2BP1 in the pre-miR-196b transfected cells determined by western blot was down-regulated in comparison to pre-miR negative control transfected cells (pre-miR CTL-) or untransfected cells (Cells), 24, 48 and 72 h after transfection. Numbers correspond to the quantification of the abundance of protein of interest normalized to the abundance of α-tubulin. (D) Schematic representation of the seed region match between miR-196b and the putative IGF2BP1 3′UTR. The mutation of five nucleotides in the seed region is shown. (E) pmiRGLO luciferase reporters containing either the wild-type or the mutant (mutated) human IGF2BP1 3′UTR were co-transfected into HepG2 cells with pre-miR negative control or pre-miR-196b (50 nM) during 72 h. 72 h post-transfection, the cells were assayed using a dual luciferase assay. Firefly luciferase values were normalized to Renilla luciferase values and plotted as relative luciferase activity (means ± 1 SD, n = 8). **: significantly different from wild-type reporter (p < 0.01), ***: significantly different from pre-miR negative control transfected cells (p < 0.001).