Fig. 4

Both the HER2 CDS and 3’UTR possess oncogenic activity. a Schematic representation of the construction of HER2 mRNA elements. (B-E) T47D cells were transfected with the HER2 3’UTR, HER2 CDS, or full-length HER2. Real-time PCR (b) and western blotting (c) analyses were performed to determine HER3 mRNA and protein levels 24 or 48 h post transfection. Cell proliferation (d) and colony formation (e) were determined by CCK8 and clonogenic assays 96 h and 7 d posttransfection, respectively. All error bars represent the standard deviation. Quantitative data were generated from a minimum of three replicates. f, g T47D cells stably transfected with the HER2 3’UTR, HER2 CDS, or full-length HER2 were s.c. injected into female BALB/c-nude mice. Tumor diameters were measured every 3 d for 30 d (f, left). Representative tumor images at day 30 are shown (f, right). Immunostaining of HER3 and HER2 levels in T47D xenograft tumor sections (g). Red, HER2/HER3 as indicated; Blue, DAPI. Scale bar, 40 μm. The results are presented as means ± SD from five mice. h, i Schematic representation of the construction of HER2 mRNA elements. The start codon of the CDS and/or the seed region of the miR-125a/b binding sites were mutated as illustrated in yellow (h). T47D cells were transfected with plasmid constructs as in (H). Cell proliferation was determined by CCK8 assays 96 h post transfection. Error bars represent the standard deviation. Quantitative data were generated from a minimum of three replicates