| Objective:Breast cancer is one of the most common malignancies of women in the world. The recurrence, metastasis and multi drug resistance of breast cancer is still a difficult problem to be improved and solved. MTDH is involved in multiple biological functions,including proliferation, cell survival, evasion of apoptosis, angiogenesis, migration and invasion, metastasis, and chemoresistance. The effect of high expression of MTDH on the proliferation and taxol sensitivity of breast cancer MCF-7 cells was studied in this study.Methods:1 A model of MCF-7 cells stably overexpressing MTDH was constructed by lentivirus and its identification: A plasmid containing MTDH gene was transformed into E.coli.DH5? and amplified. Plasmid was extracted and purified before sequencing. Using lentivirus infected MCF-7 cells after successfully sequenced and the transfection efficiency was detected by the expressing of green fluorescent protein GFP. After screened with puromycin, we establish new breast cancer cell lines which stably express MTDH gene(Named for MCF-7-MTDH) and which stably transfected negative lentivirus as controls(Named for MCF-7-vector). Use RT-PCR method to detect the expression of MTDH mRNA of blank control group(MCF-7 cells), negative control group(MCF-7-vector cells) and the experimental group(MCF-7-MTDH cells) to verify the effect of transfection.2 Using MCF-7 as blank control and MCF-7-vector as negative control, CCK method was used to detect the effect of high expression of MTDH on the proliferation and the sensitivity to taxol of MCF-7 cells.3 Using MCF-7 as blank control and MCF-7-vector as negative control, the effect of MTDH expression on cell cycle and and the effect of taxol on cell cycle was detected by flow cytometry.4 Statistical analysis: For each protocol, at least three independent experiments were performed. Results were expressed as the mean ± standard error of the mean. Statistical calculations were performed with t-test, the one way analysis of variance(ANOVA) and Nonparametric test using SPSS 17.0 software. All statistical tests were considered significant at P<0.05.Results:1 A model of breast cancer cell stably overexpressing MTDH was constructed by lentivirus and its identificationMOI value detection of MCF-7 cells: The expression of green fluorescent protein GFP was observed under microscope after lentivirus infecting. The results show that the MCF-7 breast cancer cell line MOI(Multiplicity Of Infection, MOI) value is 20, which infection efficiency reached 80% and the morphology of cells had no obvious change before and after infection.A stable overexpression of MTDH in MCF-7 cells was constructed and its identification: After 72 h cells were infected with lentivirus, the expression of green fluorescent protein was observed by fluorescence microscopy, and the transfection efficiency was more than 80% in MCF-7 group and MCF-7-vector control group. Stable transfection of MCF-7-MTDH cell line and MCF-7-vector cell line was obtained by the screening of the puromycin.RT-PCR technique was used to detect the relative expression of MTDH mRNA in three different cell lines, such as MCF-7, MCF-7-vector, and MCF-7-MTDH, the results of which were 1.000±0.100, 1.013±0.169 and 2.093±0.055. Statistical analysis showed that no significant difference in MCF-7 and MCF-7-vector cell(P > 0.05). The expression of MTDH mRNA in MCF-7-MTDH cells was higher than that in MCF-7 and MCF-7-vector cells, and the difference was statistically significant(P < 0.05).2 Overexpression of MTDH affects cell proliferation and cell cycleThe MCF-7, MCF-7-vector and MCF-7-MTDH three kinds of cells were respectively as the blank control group, the negative control group and the experimental group.Cell proliferation was detected by CCK-8 method on 0h, 24 h, 48 h and 72 h. The result showed that there was no significant difference(P>0.05) between control groups, but cell proliferation was significantly promoted in MCF-7-MTDH(P<0.01), compared with control groups.Cell cycle distribution was detected by flow cytometry.The results showed that: The results showed that the proportion of G0 / G1 phase of blank control group, negative control group, the experimental group were 32.90 ± 1.48%, 34.70 ± 1.65%, 28.80 ± 1.21%; S phase ratio of the three groups were 43.40±0.95%, 40.37±3.61%, 57.60 ±2.10% separately; G2/M phase ratio of the three groups were 23.67±0.80%, 23.60±2.51% to 13.57±2.11% separately. There was no significant difference in the cell cycle distribution between control groups(P > 0.05), but the S phase of the experimental group was prolonged, but the proportion of G0/G1 phase and G2/M phase of the experimental group was decreased compared with control groups(P < 0.05).3 Effects of paclitaxel on cell proliferation and cell cycle before and after transfectedThe MCF-7, MCF-7-vector and MCF-7-MTDH three kinds of cells were respectively as the blank control group, the negative control group and the experimental group.The inhibition rate of taxol to breast cancer cell was detected by CCK method. The result showed that: the inhibition rates of 1 ug/ml taxol to cells were 63.71±0.31%, 64.15±0.17%, 45.72±0.41% in 24 hours; the inhibition rates of 1ug/ml taxol to cells were 37.46±0.17%, 37.10±0.14%, 25.90±0.31% in 48 hours; the inhibition rates of 10 ug/ml taxol to cells were 71.11±0.87%, 72.55±0.68%, 66.97±0.84% in 24 hours; the inhibition rates of 10ug/ml taxol to cells were 69.95±0.43%, 70.77±0.21%, 59.95±0.53% in 48 hours. Statistical analysis showed that there was no significant difference in the inhibition rate of taxol between the blank control group and the negative control group(P > 0.05) at the same concentration and the time of action.. The inhibition rates of 1ug/ml and 10ug/ml on MCF-7-MTDH cells in the experimental group were lower than those in the control group after 24 h and 48 h, and the difference was statistically significant(P < 0.05). There was no significant difference in the inhibition rate of 10ug/ml taxol after 24 h and 48 h in the blank control group(P > 0.05). But the inhibition rate of 48 h in the other groups was lower than that of 24 h, and the difference was statistically significant(P < 0.05).Cell cycle of each group was detected by flow cytometry. The results showed that: Taxol increased the proportion of the three groups of cells in G2/M phase and the 10ug/ml group was significantly higher than the 1ug/ml group(P < 0.05). There was no significant difference in the proportion of G2/M phase between control groups under the same drug concentration(P > 0.05). The G2/M phase ratio of MCF-7-MTDH cells was significantly lower than that of MCF-7 and MCF-7-vector cells,(P < 0.05).Conclusions:1 A model of MCF-7 cells stably overexpressing MTDH can be successfully constructed by lentivirus.2 Overexpression of MTDH promote the proliferation of MCF-7 cells.3 Overexpression of MTDH decrease the sensitivity of MCF-7 cells to taxol through decreasing the G2/M phase ratio. |
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