Figure 3

miR-375 restores trastuzumab sensitivity by directly targeting IGF1R in breast cancer cell. A. Western blot (left) and qRT-PCR (right) analyses of IGF1R expression in parental (P) and trastuzumab-resistant (R) SKBr-3 cells. The qRT-PCR data were normalized to GAPDH. B. Luciferase activity measured 24 h after co-transfection of trastuzumab-resistant SKBr-3 with pGL3 constructs containing the wild-type or mutant 3′UTR of IGF1R, an internal control vector (pGL4.73), and synthetic miR-375 mimics. Data were normalized to the luciferase activity of control (vehicle transfected) cells. C. qRT-PCR analyses of IGF1R expression in SKBr-3 cells as modified in Figure 2. Data were normalized to mock-transfected cells. D. Pearson’s correlation analysis of the relative expression level of miR-375 (normalized to U6) and IGF1R mRNA (normalized to GAPDH) as determined using qRT-PCR in 40 human breast cancer tissue samples. E. Western blot and qRT-PCR analyses of trastuzumab-resistant SKBr-3 cells infected with a lentivirus vector expressing GFP- (control) or IGF1R-specific shRNA. Data were normalized to those of GAPDH. F. MTT assays of cells described in (E) after treatment with the indicated concentration of trastuzumab for 24 h prior to analysis. G. Flow cytometry analyses of cells in (E). Cells were treated with trastuzumab (10 μg/ml or 5 μg/ml) for 24 h and then stained with Annexin V and PI. All data are represented as the mean ± SD of n = 3 replicates. *P <0.05 and **P <0.01.