Such a finding is most likely related to alterations in the fission machinery

n the miR-7 target gene PAK1 was also observed in the modified MCF-7 cell lines. Altered miR-7 and EGFR expression was continuously monitored and remained stable in the MCF-7/miR-7KD and MCF-7/HER2D16H/miR-7 cell lines for greater than 20 cell passages. These results suggest that selective suppression of miR-7 and subsequent restoration of EGFR expression occurs in response to ectopic HER2D16 expression in MCF-7 cells. We next determined the impact of altered miR-7 expression levels on breast tumor growth in a AVE-8062 colony formation assay. Consistent with previous reports expression of HER2D16 in the MCF-7 cell line had a significant impact on colony number and diameter. In concordance with its role as a tumor suppressor, reestablished miR-7 expression to generate the MCF-7/HER2D16H/ miR-7 cell line significantly reduced the number and diameter of MCF-7/ HER2D16H colonies to levels equivalent to the MCF-7/pcDNA cell line. 7 / 16 MiR-7 Suppresses HER2D16 Oncogenic Activity Fig. 2. Altered miR-7 expression regulates breast tumor cell proliferation and migration. Mir-7 expression was stably suppressed in the MCF-7 cell line to generate the MCF-7/miR-7KD cell line and a miR-7 expression plasmid was stably introduced into the MCF-7/HER2D16H cell line to generate the MCF-7/HER2D16H/miR-7 cell line. Western blot analysis of the MCF-7 vector control, MCF-7/pcDNA and the HER2D16 expressing MCF-7/HER2D16H and MCF-7/HER2D16M1 and cell lines with altered miR-7 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19684114 expression. Relative miR-7 expression levels are indicated below each lane as determined by qRT-PCR. Colony formation assay where colony number and diameter were calculated using a ColCount Colony Counter with supplied statistical software. Asterisk indicates cell lines significantly greater than MCF-7/pcDNA and MCF-7/HER2D16/miR-7 as determined by paired Student’s t test. Cell cycle analysis was performed in a Guava Easy Cyte Mini Base System with supplied statistical software. Asterisk indicates cell cycle phase significantly different from parental cell line as determined by paired Student’s t test. Cell migration was determined in an xCELLigence CIM-Plate 16 with the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19683642 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. The data represents the mean +/2 SE of at least three independent experiments. doi:10.1371/journal.pone.0114419.g002 8 / 16 MiR-7 Suppresses HER2D16 Oncogenic Activity Conversely, suppression of MCF-7 miR-7 expression to generate the MCF-7/miR7KD cell line resulted in a significant increase in colony number to levels equivalent to ectopic expression of HER2D16. We investigated the mechanistic basis of miR-7 tumor suppressor activity by performing cell cycle analysis. We observed a significant increase in cells arrested at G1 of the cell cycle with lower levels of S phase cells in the MCF-7/pcDNA and MCF-7/HER2D16/ miR-7 cell lines both with enhanced expression of miR-7. In contrast, G1 arrest is released in the MCF-7/miR-7KD and MCF-7/HER2D16 cell lines with suppressed miR-7 expression and these cells exhibit an increase in S phase cells. Cell cycle analysis suggests that miR-7 suppresses tumor cell growth by inducing a G1 arrest with a concomitant reduction in proliferating S phase cells. This result corroborates a recent study that described a similar G1 arrest when miR-7 was expressed in Chinese hamster