| Objective This research investigates the effect of mi R-27 a mimics and inhib itor on the proliferation and drug resistance of WM239 human melanoma cells mainly by building mi R-27 a mimics and inhibitor.Methods To investigate the expression of mi R-27 a in melanoma cells WM239 and the effect of mi R-27 a on its biological characteristics through transfected mi R-27 a mimics and inhibitor into WM239 cells. The mi R-27 a mimics, inhibitor and its ne gative control were transfected into WM239 cells, the efficiency of transfection was e valuated by fluorescence microscope, the expression of mi R-27 a was detected by real-t ime fluorescent quantitative PCR, the proliferation of cell was detected by MTT, the a bility of cell colon by tablet colony formation assay, cell apoptosis and cell cycle wer e analyzed by flow cytometry.The expression of tumor suppressor gene p53 and the cell cycle gene cyclin D1,drug resistance gene MDR1, genes of MMP2 and MMP9 related with invasion and metastasis, mi R-27 a target genes of Sp1, ZBTB10 and E2F7 were detected by reverse transcription polymerase chain reaction(RT-PCR), the expression of extracellular regul ated protein kinases(ERK1/2) related with cell proliferation, caspase-3 and P glycopro tein(P-gp) related with drug resistance were analysed by western blot(WB).Results The transfection efficiency in WM239 cells was found to be about 80% to 90%. The expression of mi R-27 a was markedly up-regulated in mi R-27 a mimics groupp53 and activation of caspase-3. mi R-27 a inhibits the expression of MMP2 and MMP9 which are related to invasion and metastasis. The phosphorylation level of ERK1/2 decreases, so we presume that mi R-27 a plays a role of tumor suppressor through ERK1/2 signaling pathways. In addition, mi R-27 a can inhibit the expression of MDR1 and P-gp, increase sensitivity of WM239 cells to taxol drugs. mi R-27 a also can inhibit th e expression of target gene Sp1, promote the expression of ZBTB10. Thus, we specul ate that mi R-27 a acts as tumor suppressor genes through the Sp1-ZBTB10 axis.(2-??CT value is 26.98±0.01, F=6078713.38, P<0.05), and the mi R-27 a inhibitor group showed lower expression(2-??CT value is 0.96±0.02, F=6078713.38, P=0.165), there was no statistically significant difference. The proliferation of cell was obviously inhibited in mi R-27 a mimics group, there was statistically significant difference compare with normal control group [(0.464±0.014):(0.315±0.010)](F=129.56, P<0.05). The percentage of WM239 cells in G0-G1 phase was increased[(74.83±1.46):(63.73±1.25)](F=30.3, P< 0.05), and the percentage of WM239 cells in S phase and G2-M phase were decreased[( 21.33±1.75):(63.73±1.25),(3.90±1.31):(63.73±1.25)](F=15.0, P<0.05;F=3.7, P<0.05), the apoptosis rate of cell was significantly increased in mi R-27 a mimics group compared with normal control group [(29.67±0.91)%:(1.437±0.85)%](F=530.90, P<0.01), but there was no obvious effect on cell proliferation, cell cycle and apoptosis. There was no statistically significant difference. After transfected mi R-27 a mimics, the expression of cyclin D1 was significantly inhibited, the expression of tumor suppressor gene p53 was promoted and the aspartic acid proteolytic enzyme 3(caspase- 3) molecular related with apoptosis was activated. We found that the phosphorylation level of extracellular regulated protein kinases 1/2(ERK1/2) was decreased by detecting expression changes of related signal pathways molecules. The sensitivity of WM239 cells on taxol drug was significantly enhanced after transfected mi R-27 a mimics, and the killing effect of taxol on WM239 cells was enhanced. It had to do with inhibiting the expression of drug-resistant genes MDR1 and its protein products P-gp. Mi R-27 a could inhibit the expression of MMP2 and MMP9 which were related with cell invasion and metastasis. Moreover, we found that the expression of specificity protein 1(Sp1) which was a target gene of mi R-27 a was decreased after transfected mi R-27 a mimics, but the expression of zinc finger and BTB domain containing 10(ZBTB10) was increased, while the expression of E2F7 had no obviously change by RT-PCR detection.Conclusion mi R-27 a can suppress melanoma cell proliferation, induce cell apoptosis and block cell cycle in G0-G1 phase, suggested that mi R-27 a act as a tumorsuppressor gene. Mi R-27 a inhibits proliferation of cells, blocks the cell cycle and ind uces cell apoptosis by inhibiting expression of cyclin D1, promoting the expression of... |
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