The treatment regimens were started the day after tumor inoculation

ed cells. In addition, the levels of transcripts for EMMPRIN, an inducer of MMP expression, were found to be PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19744303 increased in BPH-1, but relatively decreased in HT1080 cells. The protein levels of MT1-MMP were not changed in BPH-1 cells treated with miR-335 alone or together with ConA but demonstrated a small elevation in miR-335 stimulated HT-1080 cells treated with ConA. The distribution of MT1-MMP in HT-1080 cells was also examined by confocal microscopy. Treatment with miR-335 increased the cell surface localization of MT1-MMP as compared with cells transfected with the control miRNA sequence. This effect supports the increased level of proMMP-2 activation found in Fig 1B. Blocking the effect of miR-335 by using an inhibitor decreased the level of MT1-MMP expression, as well as surface localization. Having found miR-335 to affect differences in cell surface localization of MT1-MMP, we examined the effect of miR-335 on cell migration and proliferation. Both HT-1080 and BPH-1 cells transfected with miR-335 showed increased motility compared to cells untreated or 5 / 12 MiR-335 Oncogenic Response Associated with MT1-MMP Fig 2. The effect of miR-335 on cell surface localization of MT1-MMP as determined by confocal microscopy in HT-1080 cells. The level of cell surface MT1-MMP Y27632 dihydrochloride custom synthesis observed in control miR treated cells was reduced by treatment with a miR-335 inhibitor. In contrast, cell surface localization of MT1-MMP was increased by miR-335. The bars indicate 20 microns. doi:10.1371/journal.pone.0132026.g002 treated with the control miR in an in vitro wound healing assay. In contrast, U87GM cells, and MCF7 and MDA-MB-23 breast cancer cells did not respond to miR335 in change of rate of filling the void with cells. HT-1080 and BPH-1 cells responded to miR-335 with increased cell proliferation, HT-1080 at 72 and 96 hours post-treatment, and BPH-1 cells at 96 hours. U87GM cells demonstrated a small enhancement of cell proliferation PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740905 at 72 hrs, whereas HCT116 cells did not change their rate of proliferation and MCF7 and MDA-MB-231 cells showed a decreased rate of proliferation in response to miR335. The response to miR-335 resulting in increased cell migration and proliferation occurred in those cell lines that responded to the microRNA with increased cell surface localization of MT1-MMP. Discussion Our studies have shown that tumor cell lines that respond to miR-335 with increased cell surface MT1-MMP also demonstrated increased cell proliferation and migration. BPH-1cells also had increased expression of EMMPRIN, a protein which stimulates MMP production. 6 / 12 MiR-335 Oncogenic Response Associated with MT1-MMP Fig 3. The effect of miR-335 on cell migration of HT-1080 and BPH-1 cells in an in vitro model of wound healing. Treatment of cells with miR-335 increased the rate of cell migration observed at 4 and 8 hours after a scratch was made in a confluent layer of cells using a pipette tip. The data were quantified as Remaining Area of Wound. doi:10.1371/journal.pone.0132026.g003 Thus, the role of miR-335 in up-regulating MT1-MMP expression may lie upstream of MT1-MMP by blocking synthesis of protein transcription factors that prevent up-regulation of the MT1-MMP gene. This proposed mechanism of MT1-MMP expression regulation in our study is evident for miR-106 which targets HOXD10, a transcriptional repressor that inhibits several genes involved in cell migration and extracellular matrix remodeling, including MT1-MMP and uPAR. In contrast, miRNAs