ovary cells. Taken together our results indicate that suppression of miR-7 is both necessary and sufficient to promote cell cycle progression and significantly enhance colony formation of breast tumor cells. We have previously shown that ectopic expression of HER2D16 induces a dramatic migration/invasion phenotype in the non-invasive MCF-7 cell line. To determine if altered miR-7 expression regulates MCF-7 cell Vorapaxar migration we performed an xCELLigence migration assay on cell lines with altered miR-7 expression. Our results indicate that suppression of miR-7 expression in the MCF7/miR-7KD cell line is not sufficient to promote MCF-7 cell migration. Consistent with our previous observations HER2D16 expression in the MCF-7/ HER2D16H cell line caused a significant increase in MCF-7 cell migration. Interestingly, reestablished expression of miR-7 in the MCF-7/ HER2D16H/miR-7 cell line completely abolished cell migration and reduced the migration index to levels observed for parental MCF-7 cells. These results indicate that suppression of miR-7 is necessary but, in contrast to tumor cell proliferation, not sufficient to promote breast tumor cell migration. MiR-7 regulates multiple oncogenic pathways that influence HER2D16 driven cell proliferation and migration We investigated the impact of altered miR-7 expression on multiple reported gene targets including FAK, insulin-like growth factor 1 receptor , PAK1, and EGFR, however, in our experimental system, EGFR was the only target that was consistently altered in response to modulated miR-7 expression. We therefore determined if EGFR is the miR-7 target gene that contributes to HER2D16 oncogenic activity. Using shRNA we inhibited EGFR expression in the MCF-7/HER2D16H cell line and determined the impact of suppressed EGFR expression on HER2D16 mediated colony formation and migration. Suppression of EGFR expression in the MCF-7/HER2D16H/EGFRKD cell line essentially abolished cell migration of MCF-7/HER2D16H cells reducing their migration activity to levels similar to parental MCF-7/pcDNA and MCF-7/ 9 / 16 MiR-7 Suppresses HER2D16 Oncogenic Activity Fig. 3. MiR-7 regulates HER2D16 induced cell migration and proliferation through different oncogenic pathways. A miR-7 expression plasmid was stably introduced PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19682619 into the MCF-7/HER2D16H cell line to generate the MCF-7/HER2D16H/miR-7 cell line and EGFR was stably knocked down by shRNA to generate the MCF-7/HER2D16H/EGFRKD cell line. Altered expression of EGFR in the indicated cell lines was confirmed by western blot analysis. Cell migration was determined in an xCELLigence CIM-Plate PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19681941 16 with the RTCA DP Instrument for 48 hrs. Cell Index was calculated using the supplied RTCA Software. Asterisk indicates that MCF-7/HER2D16H is significantly greater than the other tested cell lines as determined by paired Student’s t test. Colony formation assay where colony number and diameter were calculated using a ColCount Colony Counter with supplied statistical software. Differences between MCF-7/HER2D16H and MCF-7/HER2D16H/EGFRKD were insignificant as determined by paired Student’s t test. Western blot analysis of the indicated cell lines probed for Src kinase, 
Src kinase activated phosphorylated at Y416, and Src kinase activity through phosphorylation of FAK at Y576/577. The data represents the mean +/2 SE of at least three independent experiments. doi:10.1371/journal.pone.0114419.g003 10 / 16 MiR-7 Suppresses HER2D16 Oncogenic Activity HER2D16H/miR-7 cells. Surprising