To Explore The Mechanisms Of Nrf2 Mediated Doxorubicin Resistance In Breast Cancer By Microarray

BackgroundChemotherapy is one of the most important means to cure cancer presently,which can kill or control microscopic tumor lesions.Chemotherapy is important to treatment of breast cancer.But because of generation of resistance to chemotherapy that is one of the most important reasons to lead to chemotherapy failure.Current chemotherapy regimen based on doxorubicin has become the standard of postoperative adjuvant chemotherapy in breast cancer patients.Researches show that Nrf2 signaling pathway is involved in the drug resistance of cancer.However,there is less research on association between Nrf2 and breast cancer drug resistance.So it is particularly necessary to conduct meaningful research of the correlation between Nrf2 and doxorubicin-resistant breast cancer.ObjectiveIn this study,MCF-7/ADR as an object of study and successfully transfected Nrf2-siRNA.To study the relationship between Nrf2 and the biological characteristics of breast cancer cells(proliferation andapoptosis).GeneChip was used to analyze the expression profiles of doxorubicin-resistant breast cancer cell lines of before and after Nrf2-RNAi transfection.To explore the correlation between breast cancer drugs resistance and Nrf2 and provide the direction for researching potential downstream genes of Nrf2.The research advances the understanding of breast cancer drug-resistant mechanism,increase the sensitivity of chemotherapy,improve the curative effect of chemotherapy of breast cancer.MethodsIn this study,human doxorubicin-resistant breast cancer cell line MCF-7/ADR as an object of study and successfully transfected Nrf2-siRNA,use real time PCR to detect the expression of the target gene.To detect cell proliferation by MTT method and use the flow cytometry instrument to detect cell apoptosis and cell cycle.GeneChip primeview human was used to analyze the expression profiles of breast cancer cell lines of before and after transfection.Using the STRING database and combined with the genetic diversity,screen for the more crucial gene in the process of drug resistance of breast cancer cells to doxorubicin.Results1.Real time fluorescence quantitative: the Nrf2 mRNA expression of knockout group was obviously lower than the control group and the rate of test groups knockout was 54.3%(p <0.05).2.Used MTT results show that compared with the control group,knockout groups cell proliferation is slow.MCF7/ADR cells were cultured with epirubicin concentration of 1?g/ml for 24 hours.MTT results show that knockout groups cell proliferation slow than the control group(p<0.05).MCF7/ADR cells werecultured with epirubicin concentration of 50?g/ml for 24 hours.MTT results indicate that there was no obvious change between knockout group and control group.3.The percent of cell apoptosis was determined by Flow Cytometry,we found that MCF7/ADR cells were transfected by Nrf2-siRNA,which can reverse the drug resistance of breast cancer cells to epirubicin and become more sensitive to epirubicin.Percent of cell apoptosis in the knockout group was increased dramatically compared with the control group(P<0.05).4.By comparison of the gene expression profiles of breast cancer cell lines of before and after transfection.Screening out 878 genes with significant difference,in which 341 genes up-regulated,537 down-regulated genes by GeneChip primeview human.Differentially expressed genes associated with cell proliferation,cell cycle,apoptosis,cell material and energy metabolism and signaling cytoskeleton.Upregulated genes mainly are PHLPP2 etc and down-regulated genes mainly RAC1,PHLPP2,ASNS,SAIN2 and NPR1 etc.Conclusion1.The studies showed that through silence Nrf2 gene expression,can make the MCF7/ADR cells sensitive to adriamycin,which confirms that Nrf2 is associated with adriamycin resistance of breast cancer.2.The mechanism of breast cancer drug resistance is a very complicated.In this study,the validation of these significant differences genes were screened out by gene chip,which maybe has a close relationship for doxorubicin resistance of breast cancer cells.For that need to the further experimental.