The Role Of Spindle Assemble Checkpoint Protein BubR1 In Drug Resistance To Paclitaxel

Breast cancer is one of common female malignancy, surgery combined with chemotherapy is the main treatment.Taxol (paclitaxel, PTX), can act as a strong chemotherapy drug leading to mitotic arrest and cell death, is widely used in relapsed and refractory breast cancer chemotherapy. And acquired resistance limit its clinical application, making a considerable proportion of patients did not benefit from it. BubR1 is a key spindle checkpoint (Spindle assemble checkpoint, SAC) associated protein。It plays an important role in the inverting from premetaphase to Anaphase process. SAC's main function is correct by monitoring separation of chromosomes in order to maintain the stability of chromosome number。It plays an important role in tumor development. Early experiments by our group found, SAC proteins may be related to specific acting on the mitotic phase of the efficacy of chemotherapy drugs paclitaxel on. And has been reported, some of the BubR1 mRNA expression level was significantly lower than normal tissue of the tumor, but also prone to lymph node metastasis and recurrence. Therefore, we hypothesized that BubR1 expression in tumor cells is likely associated with tumor responsiveness to paclitaxel in a certain relationship.In this study, gene expression by interfering with BubR1, BubR1 expression levels observed after reduction of breast cancer cells in paclitaxel on cell proliferation, apoptosis, migration and other aspects of chromosome stability, and to explore new possible mechanism of paclitaxel resistance.PART I Specific interference with the mitotic checkpoint protein BubR1 and Effect EvaluationObjective:1. To construct the recombinant adenovirus BubR1.2.To evaluate and confirm (Ad-BubR1-siRNA) interference effect.Methods:1. Design and Synthesis of siRNA for BubR1 sequences..2. the application of Western Blot and quantitative RT-PCR detection of BubR1 protein and mRNA expression level, evaluation of interference effects.Results:1. BubR1 for the target gene was successfully constructed the recombinant adenovirus Ad-BubR1-siRNA. 2. Construction of the adenovirus, Ad-BubR1-siRNA-2 interfere with the strongest effect. the endogenous expression level of BubR1 of MCF-7 and Hek293 cells Infected with Ad-BubR1-siRNA-2 was significantly inhibited.Conclusion:The interference adenovirus were designed and built for the target genes BubR1. Ad-BubR1-siRNA-2 can successfully inhibited the target cells suppress expression of target genes, provide a basis for further research.PARTⅡInterference with the mitotic checkpoint protein BubR1 paclitaxel on breast cancer cells of drug resistanceObjective: Understand the interference checkpoint protein BubR1 in breast cancer cell proliferation,apoptosis,cell migration,chromosome instability in the paclitaxel effect.Methods:1. cck8 cell proliferation curves were prepared; Hochest apoptosis rate were detected; plate cloning assay clone productivity2. BubR1 inhibited cell scratch assay in breast cancer cells after Taxol treatments migration changes. 3. Karyotype inhibition assay BubR1 in paclitaxel treatment of breast cancer cells under the change of chromosome instability.Results:1. Cck8, Hochest, plate cloning experiments suggest interference with the common expression of endogenous BubR1, the breast cancer cells treated with paclitaxel showed increased proliferation, apoptosis, reduced ability to increase production rate of cloning.2. Cell scratch experiment suggested that interfere with the expression of endogenous BubR1, the breast cancer cells treated with paclitaxel showed increased migration.3. Karyotype analysis interference experiments suggest the expression of endogenous BubR1, the breast cancer cells treated with paclitaxel showed increased chromosomal instability, with malignant transformation trend..Conclusion:The interference of endogenous target gene expression of BubR1 in breast cancer cells after paclitaxel showed increased proliferation, apoptosis, reduced ability to produce cloned rate, increased migration and increased chromosomal instability. Tip BubR1 levels in breast cancer cells acquired resistance to paclitaxel play an important role in the process.