The rat 3'-untranslated region (UTR) of the IGF-1 and IGF-1R genes were amplified by polymerase chain reaction (PCR) using specific primers.
The following primers were used: IGF-1R: forward 5'-TCCTTGGATCCTGAATCTGT-3', reverse 5'-ACGTTGCCTTAGCTTCAGCC-3'; IGF-1: forward 5'- GAGGAGCCTCCCGAGGAACA-3', reverse 5'-CCTAATTTTGTCCTTTTGGG-3'.
Wild-type or mutant IGF-1 and IGF-1R 3'-UTR fragments were inserted into the XbaI restriction sites of the pmirGLO luciferase vector (Promega, USA). For transfection, 293T cells were plated in 96-well plates at 1.2×105 cells per well and co-transfected with the IGF-1 or IGF-1R 3'UTR reporter plasmid and either miR-16 mimics or negative control (NC) using Opti-MEM (Sigma-Aldrich, USA) and Lipofectamine 3000 transfection reagent (Sigma-Aldrich, USA). Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA) 36 h after transfection. The optical density of the resulting solution was measured using the EPOCH2T multiplate reader (BioTek, USA).
The following primers were used: IGF-1R: forward 5'-TCCTTGGATCCTGAATCTGT-3', reverse 5'-ACGTTGCCTTAGCTTCAGCC-3'; IGF-1: forward 5'- GAGGAGCCTCCCGAGGAACA-3', reverse 5'-CCTAATTTTGTCCTTTTGGG-3'.
Wild-type or mutant IGF-1 and IGF-1R 3'-UTR fragments were inserted into the XbaI restriction sites of the pmirGLO luciferase vector (Promega, USA). For transfection, 293T cells were plated in 96-well plates at 1.2×105 cells per well and co-transfected with the IGF-1 or IGF-1R 3'UTR reporter plasmid and either miR-16 mimics or negative control (NC) using Opti-MEM (Sigma-Aldrich, USA) and Lipofectamine 3000 transfection reagent (Sigma-Aldrich, USA). Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA) 36 h after transfection. The optical density of the resulting solution was measured using the EPOCH2T multiplate reader (BioTek, USA).