Inhibition Of CENP-E Gene Expression Could Sensitize Cervix Cancer Cell To Paclitaxe

Paclitaxel is a powerful chemotherapy agent that targets the microtubule cytoskeleton, inhibits microtubule assembly, damages the dynamic equilibrium of microtubule polymerization and depolymerization leading to mitotic arrest and cell death. At present, paclitaxel is commonly used in breast and ovarian cancer treatment.however,their clinical efficacy has been hampered due to the development of drug resistance[1]. cervical cancer is not sensitive to chemotherapy in clinic, so chemotherapy only for advanced inoperable recurrence and palliative care patients.Therefore,to investigate the cause of paclitaxel resistance and to increase their sensitivity are critical issues in the medical profession. But the mechanism of paclitaxel is not very clear. It is kowned that it affects mainly the mitosis cells.Cell division is a complex process, affected by a variety of genes and proteins.so to explore proteins which impact on cell division and the relationship with paclitaxel may be an important significancefor for studying the resistance mechanism . The mitotic kinesin Centromere-associated protein E(CENP-E) is an essential mitotic kinesin that is required for efficient stable microtubule capture at kinetochores.CENP-E plays an important role in the process of cell division: not only for the chromosome movement and congression to the spindle equator plays an important role, but also may integrates mitotic spindle mechanics with mitotic checkpoint signaling[2]. Dysfunction of CENP-E in cultured human tumor cells due to microinjection of antibodies,ablation of gene expression with siRNA or antisense oligonucleotides,or expression of dominant negative mutants leads to cell cycle arrest in mitosis with misaligned chromosomes [3,4,5]. CENP-E is also found that the most current research as potential anti-mitotic drug target[29]. Besides,CENP-E mRNA is overexpressed in avariety of human tumors relative to normal adjacent tissues[6].So we ready to study the dual role of inhibiting the expression of CENP-E and paclitaxel with tumor cells, and explore the relationship between the expression of CENP-E and the sensitivity of paclitaxel with tumor cells.In this study, the expression of CENP-E was reduced by RNAi technology, then the sensitivity of paclitaxel was observed by MTT and FCM on Hela cells with low expression of CENP-E.So to explore the relationship between the CENP-E and paclitaxel in the treatment of cancer. The results showed CENP-E silencing could sensitize cervix cancer cell to paclitaxel. And the recombinant adenovirus vector for siRNA targeting CENP-E was constructed , which provided an effective tool in vivo studies.PARTⅠRNA INTERFERENCE SUPPRESSION OF CENP-E GENE EXPRESSION IN CERVICAL CANCER CELLSObjective:To observe the CENP-E gene expression changes after recombinant expression vector containing shRNA targeting CENP-E gene transfected into cervical cancer cells (hela),and to explore the influence of down-regulation of CENP-E gene expression on the mitotic checkpoint.Methods:Plasmid vctor containing shRNA targeting CENP-E gene was transfected into human cervix cancer cell line (hela).Fluorescent quantity-polymerase chain reaction (FQ—PCR),western blot and indirect immunofluorescence were used to compare the efficiency of gene silencing after 48 hours. The cell cycle were observed when cells were dealed with tubulin depolymerization agent Nocadazol 16h after silencing CENP-E.Results:pshRNA-CENP-E inhibited the expression of CENP-E mRNA and protein in hela cell. Cells with pshRNA-CENP-E plasmid were dealed with Nocadazol, G2 / M phase cells decreased significantly. Conclusion: shRNA-CENP-E can specifically inhibit the expression of CENP-E, and provide a basis for the next experiment. CENP-E gene silencing weakened Nocadazol mitotic checkpoint-dependent inhibition of cell cycle, mitotic checkpoint function may be a certain degree of injury.PARTⅡANALYSIS THE SENSITIVITY OF CANCER CELLS TO PACLITAXE1 AFTER CENP-E GENE WAS REDUCEDObjective:To Explore the CENP-E gene and Taxol synergy on the effect of tumor cells.Methods:Cells in this experiment will be divided into three groups: non-transfected group (the control group, Hela), transfected with empty plasmid pGenesil-1 group (the control group ,Hela/pGenesil-1), as well as transfected with pshRNA-CENP-E plasmid group(the experimental group, Hela/pshRNA-CENP-E).Then these cells were treated by paclitaxel with different concentrations(1,3,10,30,100,300,1000,3000nmol/L); The inhibition rates of cells were detected through MTT assay.The apoptotic rates of cells were analysed through flow cytometry.Results:The results of MTT indicated that the growth inhibition ratio was obviously increased(P<0.05) and the growth inhibition effect was dosage dependent. The results of FCM indicated that apoptotic rate of cervix cancer cell was obviously increased after silencing CENP-E (P<0.05). Conclusion:Inhibition of CENP-E gene expression could sensitize cervix cancer cell to paclitaxe1.PARTⅢCONSTRUCT ADENOVIRUS VECTOR FOR SIRNA TARGETING THE CENTROMERE PROTEIN E(CENP-E)Objective: To construct the recombinant adenovirus vector for siRNA targeting the centromere protein E(CENP-E)for the in vivo test.Methods: The siRNA sequence targeting CENP-E gene was synthesized and cloned into the shuttle plasmid PSES-HUS to from the vector PSES-HUS-CENP-EsiRNA.It was homogenously recombinated with adenovirus backbone plasmid pAdeasy-1 in Ecoli BJ5183.Then the recombinant adenovirus vector was transfected into HEK293 cells and adenovirus was packaged and amplied.Results: The shuttle plasmid determined by sequencing, the result was consistent with the interference plasmid.The recombinant plasmid released a 4.5kb fragment and about 30kb fragmentafter PacI digestion, indicating the success of homologous recombination. Then the recombinant plasmid was lined and transfected into 293 cells, fluorescent red fluorescence can be seen under the microscope. Conclusion: The recombinant adenovirus vector containing siRNA targeting CENP-E was successfully constructed,which can provide a valid tool to study the function of CENP-E and in vivo experiment.