Discussing The Relationship Between Bmi-1, Telomerase Expression And Apoptosis Under The 5-FU Intervention In Breast Cancer Cell Line MDA-MB-468 Cells

Background and ObjectivesBreast cancer, one of the diseases severely threatening women’s health, appeared a remarkable increase tendency of incidence in recent 20 years. It is not only related to activation of proto-oncogenes and mutation or deletion of tumor suppressor genes, but also associated with the imbalance between proliferation and apoptosis of tumor cells. With the rapid development of molecular biology, multiple markers have been implicated. Oncogene Bmi-1 was a biological marker recently reported. It is an important member of PcG, which is an important factor in maintaining HOX. The whole title of Bmi-1 is B-cell specific moloney leukemia virus integration site 1. It plays a vital role in oncogenesis of many tumors. Some research indicated that Bmi-1 gene could immortalize mammary epithelial cells and might be an important factor in breast cancer development.Telomerase is a RNA-proteinase composed of RNA and protein. It has reverse transcriptase activity and could synthesize telomere with itself as template to stabilize the length of telomere. So the uncontrolled growth of was closely related to the existence of telomerase. In normal condition, telomere progressively shortens with each cell division and results in induction of cell apoptosis. It has been reported that overexpression of Bmi-1 could activate transcription of hTERT and induce the activity of telomerase and lengthened cell lifespan and leading to immortalization. But little report has been reported on expression of Bmi-1 and hTERT in MDA-MB-468 cells and their relation with cell apoptosis.In this study, through the detection of expression levels of Bmi-1 and telomerase reverse transcriptase (hTERT) of oncogene in MDA-MB-468 cells in breast cancer cell line, to discuss the relationship between Bmi-1, telomerase expression and apoptosis under the 5-FU intervention in MDA-MB-468 cells in breast cancer cell line and to make an initial evaluation of its effects and significance in the cytogenesis and development, and the correlation between them.Methods1. Cell culture and groups:breast cancer cell line MDA-MB-468 was cultured routinely. As cells grew at a good level, cells were divided at random into experiment group and control group. In experiment,4 subgroups were set with 48h and 72h intervention of 4 different concentrations (0.01μg/ml,0.1μg/ml,1μg/ml,10μg/ml) of 5-FU; In control group there was only culture fluid without 5-FU.2. MTT:Using MTT to detect the effects of different concentrations of 5-FU (0.01μg/ml,0.1μg/ml,1μg/ml,10μg/ml) on the inhibition of MDA-MB-468 cell proliferation in breast cancer cell lines, and to determine the experiment concentrations of the drug.3. Flow cytometry and TUNEL:To detect the apoptosis situation under the intervention of 1μg/ml, 10μg/ml 5-FU on MDA-MB-468 cells after 48h,72h.4. RT-PCR:Using the RT-PCR to detect the mRNA expression of oncogene Bmi-1 and hTERT under the intervention of 1μg/ml,10μg/ml 5-Fu on MDA-MB-468 cells after 48h,72h and comparing to the control group to analyze the relationship between Bmi-1, hTERT-mRNA expression and their correlation with apoptosis. 5. Western Blot:Using Western Blot to detect the protein expression of oncogene Bmi-1 and hTERT under the intervention of 1μg/ml, 10μg/ml 5-Fu on MDA-MB-468 cells after 48h,72h and comparing to the control group to analyze the relationship between Bmi-1, hTERT-protein expression and their correlation with apoptosis.6. Statistical analysis:SPSS 13.0 software package was applied in statistical analysis, P<0.05 as statistically significant. Spearman level correlation analysis was applied in relations between two variables. a=0.05 indicates statisitical significance.Results1. The results of MTT:5-FU intervention on MDA-MB-468 cells could significantly inhibited growth in all experimental groups. The differences of OD value between the groups were statistically significant (P<0.05), and the dose and time dependent existed in a certain concentration.2. The results of TUNEL:under the intervention of 1μg/ml,10μg/ml 5-Fu on MDA-MB-468 cells after 48h,72h, the apoptosis index increased. The differences between the groups were significant (P<0.05).3. The results of flow cytometry:under the intervention of 1μg/ml,10μg/ml 5-Fu on MDA-MB-468 cells after 48h,72h, the apoptosis rate increased. The differences between the groups were significant (P<0.05).4. The results of RT-PCR:under the intervention of 1μg/ml, 10μg/ml 5-Fu on MDA-MB-468 cells after 48h,72h, the mRNA expression levels of Bmi-1 and hTERT gradually decreased. The expression of Bmi-1 and hTERT-mRNA between groups were significant differences (P<0.05).5. The results of Western Blot:under the intervention of 1μg/ml,10μg/ml 5-Fu on MDA-MB-468 cells after 48h,72h, the protein expression of Bmi-1 and hTERT gradually decreased. The expression of Bmi-1 and hTERT between groups were significant differences (P<0.05).6. The expression of Bmi-1 and hTERT in MDA-MB-468 cells showed a positive correlation through the statistical analysis (P<0.05).Conclusion1. With intervention of 5-FU, the mRNA and protein expression of Bmi-1 and hTERT in MDA-MB-468 cells decreased in time and concentration dependent manners.2.5-FU may accelerates the apoptosis of MDA-MB-468 cells by down-regulating the expression of Bmi-1 and hTERT.