Figure 2.

MiR-4458 targets 3’-UTR of IGF1R to inhibit its expression. A. Bioinformatics analysis showed that miR-4458 binds to 3’-UTR of IGF1R mRNA. B. We modified miR-4458 levels in a human disc cell line, HNPSV. We transfected HNPSV cells with either miR-4458 or antisense for miR-4458 (as-miR-4458). HNPSV cells were also transfected with a null sequence as a control (null). Modulation of miR-4458 levels in HNPSV cells was confirmed by RT-qPCR. C. The intact 3’-UTR of IGF1R mRNA (IGF1R 3’-UTR), together with a 3’-UTR with mutant at miR-4458-binding site of IGF1R mRNA (IGF1R 3’-UTR mut), was then cloned into luciferase reporter plasmids, and used for co-transfection with miR-4458-modified plasmids into HNPSV cells. First, HNPSV cells were co-transfected with 1 μg as-miR-4458/null plasmids and 1 μg IGF1R 3’-UTR plasmids, showing that miR-4458 depletion significantly increased luciferase activities. Next, HNPSV cells were co-transfected with 1 μg miR-4458/null plasmids and 1 μg IGF1R 3’-UTR or IGF1R 3’-UTR mut plasmids, showing that that IGF1R 3’-UTR plus miR-4458 had the most repression for IGF1R, and the IGF1R 3’-UTR mut failed to decrease luciferase activities by miR-4458. *p<0.05. N=5.