Fig. 5
Effect of the HER2 3’UTR on trastuzumab resistance. a Copies-per-cell analysis of miR-125a/b, HER2, and HER3 mRNA in HER2-overexpressing cells. b, c Real-time PCR analysis of HER2 and HER3 mRNA levels in AU565 cells treated with the indicated concentration of trastuzumab (b) or 10 μg/ml trastuzumab at the indicated times (c). EGFR served as a negative control. **, P < 0.01 compared to the control. ***, P < 0.001 compared to the control. d Real-time PCR analysis of HER3 mRNA levels in AU565 cells treated with the indicated concentration of trastuzumab with or without miR-125a/b inhibitors. e Sequencing of the HER2 and HER3 genes in wild type (MREWT) and CRISPR/Cas9-mutated (ΔMRE) miR-125a/b response elements. Boxes in red indicate the seed region of the miR-125a/b binding sites. f, g Real-time PCR (f) and FACS (g) analyses of HER3 mRNA and protein levels in AU565 WT and ΔMRE cell lines. h Cell proliferation was determined by CCK8 assays. i AU565 WT and ΔMRE cells were treated with 10 μg/ml trastuzumab or control IgG at the indicated times. The inhibition rate of cell proliferation was calculated as: Inhibition rate (%) = [(cell proliferation without trastuzumab–cell proliferation with trastuzumab)/cell proliferation without trastuzumab] × 100%. j T47D cells stably transfected with the HER2 3’UTR, HER2 CDS, full-length HER2, or control vector were s.c. injected into female BALB/c-nude mice. When the xenograft tumors grew to a volume of 0.1 cm3, mice were treated with trastuzumab (10 mg/kg). Tumor diameters were measured every 5 d for 20 d (j, left). Representative tumor images at day 20 are shown (j, right). The results are presented as means ± SD from five mice