The Effect Of5-FU On Expression Of Bmi-1, Sca-1and Oct-4in Human Breast Cancer Cell Line MDA-MB-468

Background and objectives:Breast cancer is the commonest malignancy in female, leading to extraordinary somatic and psychological harmness to women. Surgery assisted with chemotherapy and radiotherapy is the common measure in modern treatment of breast cancer, these measures have improved the prognosis of patients with breast cancer greatly. However, therapy resistance become a serious obstacle in the treatment, drug resistance in chemotherapy of cancer cells make the tumor easy to recur and difficult to cure. In the recent view of caner stem cell hypothesis, cancer stem cells retain the properties of stem cells as self-renewal and potential multilineage differention and play an important role in the initiation, progression, invasion, metastasis, drug resistance, relapse of tumors.Recently, relations between breast cancer stem cells and drug resistance in chemotherapy became a hot research topic.Bmi-1gene, an important member of the polycomb gene family, presents functions as transcription inhibition and maintaining self-renewal of stem cells. Bmi-1is probably a marker for cancer stem cells in breast cancer. A Bmi-1high expression level has been seen in many malignacies including breast cancer in both domestic and foreign reports; but little review has been found on the relation between Bmi-1 expression and cancer stem cells. Sca-1and Oct-4genes are of importance in maintaining activity and biological features of stem cells and associated with tumor stem/progenitor cells, these two were selected as markers of cancer stem cells in breast cancer cell line.In this research, RT-PCR and Western blot techniques were employed to detect the expression of Bmi-1and Sca-1, Oct-4before and after the interference of5-FU in human breast cancer cell line MDA-MB-468, in order to further understand the relation of Bmi-1expression with breast cancer stem cells and drug resistance in breast cancer, find a novel molecular treatment target and improve the efficacy of chemotherapy of breast cancer.Methods1. MTT method was applied to detect the inhibition effect on proliferation of different concentrations of5-FU to MDA-MB-468cells with48h and72culture, a curve of proliferation inhibition rate was drawn, and a suitable experiment concentration of5-FU was chosen.2. MDA-MB-468cells was serially passaged under continuous interference with the suitable concentration of5-FU,6generations of cells were collected; cells with5-FU interference were designed as experiment group and a corresponding control group with no5-FU was set, the cells of both group were serially passaged,6generations of cells were collected.3. RT-PCR and Western blot were employed to examine mRNA and protein expression of Bmi-1and Sca-1, Oct-4in the6generations of cells of both control group and experiment group.4. Statistical analysis:SPSS13.0statisitical software was used to analyze the data in this experiment. One-way ANOVA was used in comparison of mRNA and protein expression levels of Bmi-1and Sca-1, Oct-4in cells of different generations, Pearson correlation analysis was used to analyze the relation among these three markers. α=0.05indicates statisitical significance. Results:1. Results of the MTT test showed that5-FU could inhibition the proliferation of human breast cancer cell line MDA-MB-468, the differences between the groups with different5-FU concentrations were of statistical significance (P<0.05), the5-FU concentration of0.1μg/mL was choosed as the suitable experiment concentration.2. Experiment data from RT-PCR tests showed that the differences between the relative mRNA expression values of Bmi-1and Sca-1, Oct-4(two factors on behalf the quantity of cancer stem cells) in the6generations of MDA-MB-468cells were not statistically significant in control group (all P>0.05) and that the differences between the relative mRNA expression values of Bmi-1and Sca-1, Oct-4in the6generations of MDA-MB-468cells were of statistical significance in experiment group cells (all P<0.05); the mRNA expression of Bmi-1, Sca-1and Oct-4showed the following tendency in the6generations of passaged cells: decrease (1st generation)-increase (2nd generation)-continuous increase (3rd generation)-decrease again (4th generation)-increase once more (5th generation)-decrease(6th generation).3. Western blot tests indicated that the differences between the relative protein expression values of Bmi-1and Sca-1, Oct-4in the6generations of MDA-MB-468cells were not statistically significant in control group (all P>0.05) and that the differences between the relative protein expression values of Bmi-1and Sca-1, Oct-4in the6generations of MDA-MB-468cells were of statistical significance in experiment group cells (all P<0.05); the proteion expression of Bmi-1, Sca-1and Oct-4showed the following tendency in the6generations of passaged cells similar to the mRNA expression tendency:decrease (1st generation)-increase (2nd generation)-continuous increase (3rd generation)-decrease again (4th generation)-increase once more (5th generation)-decrease(6th generation).4. The expression of Bmi-1was positively correlated with stem cell associated factors Sca-1、Oct-4(r≈1, all P<0.01).Conclusions:1. The expression of Bmi-1gene is positively correlated with those of stem cell associated factors Sca-1and Oct-4in breast cancer cell line MDA-MB-468cell, Bmi-1may be a novel marker for cancer stem cells in breast cencer.2. Adminatration of5-FU can affect the expression level of Bmi-1and ration of cancer stem cells in breast cancer cell line MDA-MB-468, Bmi-1gene may be associated with drug resistance to chemotherapy and recurrence in breast cancer.