Figure 4

Establishment of IGF1R as a target of mir-376a/c. (A) The 3'UTR of the IGF1R gene showing potential binding sites for mir-376a/c (marked in grey). The sequence conservation from human to lizard in the vicinity of mir-376c and 376a seed sequences and the putative interaction of the miRNAs and the mRNA are shown (taken from TargetScan at http://www.targetscan.org/). (B) IGF1R mRNA and protein levels in NHEM and several melanoma cell lines using qRT-PCR and Western blot, respectively. (C) IGF1R mRNA and protein levels in pTER-transfected and mir-376a/c-transfected mel33B1 melanoma cells using qRT-PCR and Western blot, respectively. (D) Melanoma cell lines stably transfected with pTER, mir-376a or mir-376c were transiently transfected with a control-luciferase vector or with a vector containing luciferase attached to an element of the 3'UTR of the IGF1R gene containing the putative binding sited for mir-376a/c (designated IGF1R 3'A). The ratio of expression of luciferase/renilla was normalized relative to control-transfected cells for each of the cell lines. * signifies p < 0.05 using two-way ANOVA. (E) The migration of control pTER-transfected melanoma cells was assessed in the absence and in the presence of varying doses of AG-1024 using the in-vitro transwell method. Representative micrographs of the transwell membrane are seen. This experiment was repeated three times.