Study Of The Relationship Between Topoismerase And The Prognostic, MDR Of Ovarian Cancer

Ovarian cancer is a malignancy tumor which seriously threats women's lives and health. In recent 30 years,Ovarian cancer's 5 year survival rate is long remained between 30-50%.Chemotherapy has no effect on its total survival.It is mainly duo to cancer cell's endurance for chemotherapy,topoismerase is a criticality intranuclear endoenzyme which regulate DNA spatial configuration dynamic state,there is correlation between the expression of topoismerase,but the concrete mechanism need further study.So our study discussed the the relationship between topoismerase and the survival,MDR of ovarian cancer at first,then construct RNA expression vector to study its biological behaviour,expect to find out direct evidence that topoismerase has correlation with ovarian cancer's multidrug resistance.The expression of topoismerase and its relationship with clinic pathology and prognosticObjective:study of the relationship between topoismerase and ovarian cancer's prognostic and MDR.Materials and methods:Detect the mRNA expression of TopoⅡα,TopoⅡβand Topoâ… in 47 ovarian cancer,20 innocence ovarian tumour,20 normal ovarian tissues by RT-PCR, compared the positive ratio and semiquantitative,analyze the results with clinic pathology by statistics,and use single-factor analysis of the survival and Cox regression analysis.Results:TopoⅡαis high expressed in malignancy ovarian tumor tissues and recurrence ovarian cancer tissues;TopoⅡβis high expressed in malignancy ovarian tumor tissues,and has correlation with metastasis;TopoⅡαis a independence prognostic factor.Conclusions:topoismerase has closet correlation with ovarian cancer's prognosis,could be considered as an index to judge the ovarian cancer's prognosis and drug resistance.The construct of topoismeraseⅡαRNA expression vectorObjective:construct TopoⅡαRNA expression vector.Materials and methods:acquire 4 pairs of siRNA by chemosynthesis,select one pair which silence the TopoⅡαgene best by RT-PCR,according to the psilencer4.1's designing requirement,synthesis double strands DNA,also a pair of negative control.By annealing, double enzyme cutting,connecting,transformation,select the positive clones,after successful sequencing identification,transfection SKOV3/DDP which high expresses TopoⅡαby Lipofectamine2000,screen single clone cell which expresses recombinant plasmid by cytotoxicity test.Results:acquired 3 cell lines which express psilencer4.1-TopoⅡα,psilencer4.1-negtive control,empty psilencer4.1 plasmid respectively.Conclusions:the success of the construction of RNA expression vector establishes foundation for further gene function study.The vitro experiment of RNA interference topoismeraseⅡαand reversal its drug resistanceObjective:study the relationship between TopoⅡαand ovarian cancer's drug resistance,and the possible mechanisms.Materials and methods:The experiment includes transient transfection and stable transfection RNA interference.The silence effect is detected by RT-PCR and Western Blot;MTT to detect the resistance index(RI);HPLC(high performance liquid chromatography)to detect the drug concentration in the cells;flow cytometry to detect the cells' DNA cycle.Results:It could silence the objective gene's expression by transient and stable RNAi.After RNAi,the AI and the drug concentration in the cells are reduced,the ratio of G₀-M stage in the cells is increased and S stage is decreased.Conclusions:we acquire direct evidences that DNA topoismerase have correlation with ovarian cancer.Confirmed the clinical observations that DNA topoismerase plays an important role in tumour cell's multidrug resistance.