Livin Gene Expression In Ovarian Carcinoma And The Effect Of It's Silencing On Ovarian Carcinoma Cell Development And Sensitivity To Chemotherapy

Introduction and objectivesThere are 60%-90% epithelial ovarian tumor in ovarian primary malignant tumors. The cytoreductive surgery and cisplatin-based chemotherapy is the basic treatment principle. But the tumor cells show drug resistance which let many patients' situation be serious or relapse new tumor and lead to the five-year-survival be only 20%-30%. The morbidity of ovarian neoplasms is in the rank of the third in female genital malignant tumor, but the first mortality. Many researches have confirmed the fact that it is very important that increased resistance to apoptosis be in tumor occurrence and development. Moreover, apoptosis defficiency is considered to be a major cause of the therapeutic resistance of tumors in the clinic, since many chemotherapeutic agents act through the induction of apoptosis and become a hallmark of many tumor cells. It is therefore hoped that an increased understanding of the regulatory circuits contributing to the apoptosis resistance of cancer cells may provide a rational basis for the development of novel therapeutic strategies by specifically interfering with the activity of antiapoptotic factors in tumor cells.The inhibitor of apoptosis protein (IAP) family consists of a group of structurally related proteins with antiapoptotic properties. A novel member of this family is the Livin protein . It has been shown that ectopic expression of Livin can block apoptosis induction by a variety of proapoptotic stimuli by inhibiting caspases-3,-7and -9. The livin gene exhibits a restricted expression pattern, since it has been found to be expressed in certain tumor cells, including melanoma or HeLa cervical carcinoma cells, but not, or to substantially lesser amounts in most normal adult tissues. Its expression in tumor cells suggests that Livin,as it is assumed for other IAPs,may contribute to tumorigenesis by causing apoptosis resistance. Consequently, inhibition of Livin expression may represent an interesting therapeutic strategy, by reduction of Livin gene leading to relieve the inhibition to apoptosis, and subsequently promote tumor cells to die and enhance sensitivity of tumor cells to drug.RNA interference (RNAi) is a kind of specific gene silence post transcription, it can lead to the sequence-specific degradation of endogenous RNAs that match the double- stranded RNA (dsRNA) by introducing the dsRNA into cells. As a new technique, RNAi shows a good prospect as it is simple, quick, efficient and economical. There is not report of studying the function of Livin in ovarian carcinoma by RNAi in foreign country and in interior. In this experiment, we block the Livin gene with RNAi and discuss the change of ovarian carcinoma cells on proliferation, apoptosis and sensitivity to cisplatin. We hope we can get more knowledge about the function of Livin in ovarian carcinoma occurrence and development, and then research the way of gene therapy. It includes three parts in our report. The first part, Title: The expression and significance of Livin and SMAC in ovarian epithelial carcinoma tissues. Objective: The aim of this study is to detect the expression and correlation of Livin and SMAC in normal ovarian tissues and ovarian tumor tissues and to explore their clinical significant. The second part, Title: Construction and identification of lentiviral vector expressing of shRNA targeting Livin gene. Objective: To construct a recombinet lentivirus vector expressing short hairpin interference RNA (shRNA)of Livin gene, present the basis for further studying the anti-apoptosis activity and gene therapy in target tumor. The third part, Title: Research on the Effect of Silencing Livin gene on ovarian carcinoma cell development and sensitivity to chemotherapy. Objective: To investigate the influence on ovarian carcinoma cells proliferation and apoptosis and the change of chemosensitivity to cisplatin after silencing of Livin gene by special siRNA meditated by recombinet lentivirus in SKOV-3 cell line. MethodsThe first part: The expression of Livin mRNA and protein in 20 cases of normal ovarian tissues, 20 cases of benign tumor and 50 cases of ovarian epithelial malignant tumor were detected by using RT-PCR( reverse-transcription PCR) and western blotting. Immunohistochemistry SABC method and image analysis system were using to observe the expression of Livin and SMAC in the same tissues. The relationship between expression of Livin, SMAC, and ovarian tissue type as well as the tumor grade and clinical stage of EOC patient was evaluated by SPSS software.The second part: Four shRNA sequences targeting human Livin gene were designed ,after synthesis and annealing , and were cloned into the pGCL-GFP plasmid. Livin sequence was amplified from plasmid which contained livin gene definitely by PCR and subcloned into the pdsRED2-N1-3FLAG plasmid. The two recombinant plasmids were cotransfected 293T cells with the help of lipfectamine2000 to observe the interference efficiency by western-blot and fluorescent microscope test .The most efficient plasmid was packed to construct recombinant lentivirus vector expression shRNA of Livin .The changes of Livin mRNA was detected by real-time RT-PCR in SKOV-3 post infection.The third part: The proliferation ability of ovarian carcinoma cells was test by MTT experiment. The apoptosis percent of sequence specific siRNA were assessed with flowcytometry and the change of Caspase-3 was detected by western blotting. The chemosensitivity of transfected cells to cisplatin was measured by MTT. The apoptosis percent was tested after Livin siRNA plus cisplatin on SKOV-3 by Annexin-V-PE/7AAD and by western blotting to measured the Caspase-3.ResultsThe first part: In normal tissues, benign tumor and malignant ovarian epithelial tumor, the positivity of Livin is 10%, 15% and 64% respectively. The difference among three groups is significantly. The expression of Livin in malignant tumor is much higher than in normal tissues and benign tumor (P <0.01 ). The level both mRNA and protein of two isoforms were same and express in the same time of the same positive tissue. In ovarian epithelial carcinoma, there is a positive correlation between the expression of Livin and the tumor grade and does not related to tumor stage;The expression of SMAC are negative related to tumor stage and tumor grade.The second part: The recombined vectors of four RNAi and one overexpression Livin were identified by sequencing to be correct . The expression of Livin protein were blocked nearly 75% and 80% respectively by the two efficient plasmid in target cells and are dependent with dose. The lentivirus was produced successfully by 293T cells with titer of 5×106 TU/ml.The titer could reach 3×108TU/ml if concentrating was carried out. The most suitable MOI (multiplicity of infection) of lentivirus for SKOV-3 cells was 20. When SKOV-3 cells were infected by recombined lentivirus, the levels of mRNA encoding Livin in cells was reduced by 70%.The third part:MTT results show that compare with nonspecific and control groups, specific infection with Livin siRNA lentivirus group's value of absorbing light is significant at moment of, 72h and 96h (P <0.05) .Flowcytometry results show that the apoptosis of specific group's SKOV-3 cells obviously increase with Livin expression decrease and three times than the other groups (P<0.01) . No significant difference in apoptosis rate is founded between the control group and nonspecific group. The content of Caspase-3 active form of specific group is higer than in the other groups and there is no significant difference between the other groups. After three groups expose to cisplatin,on the same conditions, cmpare with the other groups the specific group' apoptosis rate is higher and the survival rate is lower and the content of Caspase-3 active form is higer more obviously.ConclusionThe first part: The increased mRNA and protein expression of Livin and depressed protein expression of SMAC play important roles in the carcinogenesis and differentiation of ovarian epithelial carcinoma; These foundings suggest that Livin could be one of the useful targets for treatment of ovarian carcinoma.The second part: We had successfully constructed shRNA-lentivirus which could suppress Livin mRNA and protein expression in SKOV-3 cells effectively and specifically in dose-dependent and time-dependent manmer, this provided a suitable experimental tool for exploring Livin function and mechanism furthermor.The third part: Livin siRNA can inhibit SKOV-3 cell proliferation, induce cell apoptosis and significantly. Specific siRNA inhibit cell proliferation and induce cell apoptosis maybe relate with it can inhibit the expression of Livin gene and increase the content of Caspae-3. Livin siRNA can induce cell apoptosis significantly and increase the sensitivity of SKOV-3 to cisplatin.