Figure 2

MiR-497 targets IGF1-R. (a) A schematic illustration of base paring between miR-497 (upper) and miR-424 (lower) and the 3′UTR of IGF1-R. Substitution of three consecutive bases (UGC to ACG) at the 3′UTR of IGF1-R for the mutant reporter construct is also shown. (b) A schematic illustration of the pSI-CHECK2-based luciferase reporter constructs used for examining the effect of miR-424 and miR-497 on the 3′UTR of IGF1-R. (c) HCT116 cells were co-transfected with the indicated reporter constructs and Renilla luciferase plasmids. Twenty-four hours later, the reporter activity was measured using luciferase assays. The data shown are the mean±s.e. of three individual experiments. (d) Left panel: HCT116 cells were co-transfected with the indicated reporter constructs and Renilla luciferase plasmids. Scrambled, anti-miR-424 or anti-miR-497 oligonucleotides were also co-transfected. Twenty-four hours later, the reporter activity was measured using luciferase assays. Right Panel: qRT–PCR analysis of miR-424 and miR-497 in total RNA from HCT116 cells transfected with scrambled, anti-miR-424 or anti-miR-497 oligonucleotides. The data shown are the mean±s.e. of three individual experiments. (e) Left panel: HCT116 cells were co-transfected with the indicated reporter constructs and Renilla luciferase plasmids. Scrambled, miR-195 mimics or miR-497 mimics were also co-transfected. Twenty-four hours later, the reporter activity was measured using luciferase assays. Right Panel: qRT–PCR analysis of miR-195 and miR-497 in total RNA from HCT116 cells transfected with scrambled, miR-195 mimics or miR-497 mimics. The data shown are the mean±s.e. of three individual experiments. (f) Upper panel: HCT116 cells were transfected with scrambled, miR-195 mimics or miR-497 mimics. Twenty-four hours later, whole cell lysates were subjected to western blot analysis of IGF1-R and GAPDH (as a loading control). Lower panel: HCT116 cells were transfected with scrambled, anti-miR-424 or anti-miR-497 oligonucleotides. Twenty-four hours later, whole cell lysates were subjected to western blot analysis of IGF1-R and GAPDH (as a loading control). The data shown are representative of three individual western blot analyses. (g) Upper panel: western blot analysis of IGF1-R in whole cell lysates from CRC tissues that expressed high levels of miR-424 but different relative (low or high) levels of miR-497 sampled from CRC tissues shown in Figure 1c. Lower panel: western blot analysis of IGF1-R in whole cell lysates from normal colon mucosa. Whole cell lysates from paired colon cancer tissue samples as shown in Figure 1c were included as controls. The data shown are representative of three individual western blot analyses.