Fig. 4

Characterization of cells transfected with miR-302a-AMO and the CUL1 expression plasmid. After transfected miR-302a-AMO or the pcDNA3.1-CUL1 plasmid in HCC-1937 and MDA-MB-231 cells for 6 h, HCC-1937 and MDA-MB-231 cells were exposed on Selumetinib (10 μM). The CUL1 mRNA (a) and protein (b) expression levels were detected by qRT-PCR and western blot analysis, respectively. The CUL-1 level on both mRNA and protein increased significantly in miR-302a-AMO and pcDNA3.1-CUL1 group (For HCC1973 cells, the CUL1 levels were 0.97 ± 0.08, 3.81 ± 1.17 and 14.18 ± 3.56 respectively. For MDA-MB-231 cells, the CUL1 levels were 0.95 ± 0.15, 3.86 ± 0.77 and 17.44 ± 4.71 respectively. *P < 0.01 compared with the Selumetinib control group, which is 1). c Cell viability was detected by MTT method after single or combined treatment with Selumetinib and miR-302a-AMO or the pcDNA3.1-CUL1 plasmid (For HCC1973 cells, the inhibition ratio were 53.72 ± 8.34 vs 55.07 ± 7.83, 38.17 ± 4.89 and 36.73 ± 6.11 respectively. For MDA-MB-231 cells, the inhibition ratio were 54.83 ± 7.81 vs 55.33 ± 5.43, 41.11 ± 5.47 and 37.27 ± 4.25 respectively. *P < 0.01 compared with the Selumetinib control group). d Change in the apoptotic ratio of was HCC-1937 and MDA-MB-231 cells were detected by FACS (For HCC1973 cells, the living cell ratio were 55.07 ± 7.61 vs 52.33 ± 11.52, 73.27 ± 7.26 and 68.03 ± 10.95 respectively. For MDA-MB-231 cells, the living cell ratio were 55.37 ± 5.51 vs 50.53 ± 11.91, 76.43 ± 10.21 and 71.82 ± 14.07 respectively. *P < 0.01 compared with the Selumetinib control group). e As soon as the same treatment on HCC-1937 and MDA-MB-231 cells, the cell cycle arrest was in line with apoptosis trends ((For HCC1973 cells, the G1 ratios are 69.53 ± 5.35 vs 69.02 ± 7.28, 62.33 ± 8.27 and 60.86 ± 10.69 respectively. For MDA-MB-231 cells, the G1 ratios were 72.53 ± 2.91 vs 72.94 ± 7.55, 62.53 ± 9.33 and 60.56 ± 9.33 respectively. *P < 0.01 compared with the Selumetinib control group). f Another apoptosis detected method, tunnel, produced the identical results. g and h indicated the negative effect of Selumetinib on migration of HCC-1937 and MDA-MB-231 were reverse by miR-302a-AMO and CUL1 over-expression plasmid