ed COP1 expression in control EC. doi:10.1371/journal.pone.0137361.g004 miR-214. Moreover, mutant 1 and 2 vectors, which contained a mutated target sequence complementary to the miR-214 seed region, relieved the repressive effect of miR-214 on the luciferase activity. To investigate whether miR-214 was able to regulate COP1 expression, we Tipifarnib web conducted mRNA qRT-PCR and immunoblotting for determining the expression of COP1 mRNA and protein in miR-214-transfected HSA cell lines. Our data showed that the ectopic expression of 10 / 19 miR-214 Is a Noble Anti-Oncomir in Canine Hemangiosarcoma Fig 5. COP1 was overexpressed in HSA cell lines. The protein expression of COP1 in HSA cell lines and normal EC assessed by immunoblotting. COP1 was strongly expressed in HSA cell lines, however, normal EC weakly expressed COP1. doi:10.1371/journal.pone.0137361.g005 miR-214 down-regulated COP1 expression at both mRNA and protein levels. Furthermore, miR-214-knockdown by the inhibitor up-regulated COP1 expression in normal EC, suggesting that miR-214 is a key regulator of COP1. Taken together, these results demonstrated that PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19736355 miR-214 directly targeted COP1 mRNA through binding to the predicted seed sequence in the 3’UTR of COP1 mRNA and thereby negatively regulated COP1 expression at both mRNA and protein levels. COP1 was overexpressed in HSA cell lines Because miR-214 directly regulated COP1 expression, we hypothesized that down-regulation of miR-214 induced up-regulation of COP1 in HSA. In order to validate the differences of COP1 expression, we assessed the expression of COP1 protein in the HSA cell lines and control EC by immunoblotting. Consistent with our hypothesis, COP1 protein was overexpressed in HSA cell lines, whereas normal EC weakly expressed COP1. The result implies that miR-214 down-regulation possibly leads to COP1 overexpression in HSA; therefore, miR214-dependent COP1 overexpression is a key event for dysregulated p53 expression in HSA patients. COP1 knockdown induced apoptosis and up-regulation of p53-regulated genes Our results showed that miR-214 induced apoptosis and that miR-214 directly targeted COP1, which was overexpressed in HSA cell lines. Next, to confirm whether COP1 knockdown could result in apoptosis in HSA cells, we conducted specific knockdown of COP1 by using its siRNA. The results indicated that COP1 knockdown decreased the number of viable cells, but increased the number of apoptotic cells and up-regulated the expression of the p53-regulated genes in the HSA cell lines. However, siR-cop1 induced slight growth inhibition without apoptosis or significant activation of p53-regulated genes except BAX in control EC similar to miR-214-transfection in control EC. Thus, COP1 knockdown induced apoptosis in HSA cells by up-regulating p53-regulated gene PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19736794 expression, similar to the results obtained using miR-214-transfection. These results suggest that COP1 was an essential molecule responsible for miR-214-mediated apoptosis in HSA cells. 11 / 19 miR-214 Is a Noble Anti-Oncomir in Canine Hemangiosarcoma Fig 6. COP1 knockdown decreased the number of viable cells and induced apoptosis in HSA cell lines just as did miR-214-transfection. Cell viability was assessed by performing the MTT assay. COP1 knockdown by siRNA dose-dependently decreased the number of viable cells of all HSA cell lines. COP1 knockdown decreased the viable cells of control EC; however, the degree was slight compared to that of HSA cell lines. All data are presented a