IGF-1R was directly targeted by miR-223.

<p>(A) Quantitative PCR results of IGF-1R. Two pairs of primers (IGF-1R-1 and IGF-1R-2) designed at different positions of IGF-1R produced similar results showing suppression caused by miR-223. (B) IGF-1R protein level was suppressed by miR-223. Both precursor and mature IGF-1R (2 bands) were suppressed after miR-223 overexpression. The phosphorylation level was subsequently down regulated in agreement with the total protein level of IGF-1R. The suppression was abolished by transfection of the cells with IGF-1R cDNA without 3′UTR. (C) (D) The protein bands were scanned to measure the integral density in B experiments and normalized to those of GAPDH. The relative expression was then compared. Experiments were carried out 3 independent times. *<i>p</i><0.05, **<i>p</i><0.01 (E) The top panel showed the structure and cloning site of psi-<i>CHECK</i>™-2 vector. Wild type and three nucleotides mutated within IGF-1R 3′UTR were cloned into the reporter based on the predicted binding site in the 3′UTR of IGF-1R with miR-223 seed sequence. The right panel showed that sequences of either wild type (upper) or mutant (down) were confirmed by sequencing. (F) Dual Luciferase experiment of IGF-1R 3′UTR was conducted. The expression of the reporter containing IGF-1R 3′UTR was suppressed by miR-223, but not in the mutated construct. Inhibition of miR-223 abolished the suppression of miR-223 on IGF-1R 3′UTR at a final concentration of 50 nM. **<i>p</i><0.01 (G) Growth curve was measured by CCK-8 assay and the results indicated that the suppression of miR-223 group could be overcome by re-expression of IGF-1R. The group transfected with pcDNA 3.1-His/Myc served as the control. **<i>p</i><0.01 (H): Immunohistochemical staining of IGF-1R in the sections. IGF-1R was labeled in red carried by secondary antibody and the signal was stronger in EV group. In miR-223 group, the signal of IGF-1R staining was significantly weaker than that in EV group. In blank group, PBS replaced the first antibody in either EV or miR-223 group. Original magnification 10 ×.