Silencing Of E2F3 Expression Using RNAI With AAV Virus Expressing Vector Suppresses Growth Of Bladder Cancer 5637 Cell Line

Bladder cancer is the most common urological malignant tumor, more common in developed countries and regions, but also one of the most common malignant tumor in our clinical and a direct threat to the survival. The most common pathological type of bladder cancer is transitional cell carcinoma. Its biological features include:multi-focal, multi-gene participation, multi-phase heterogeneous growth, easy to relapse and metastasis. In recent years, bladder tumor's incidence is on the rise, and easy to relapse, and easily transformed into invasive cancer. Operation especially transurethral resection of bladder tumor has been the main measure in treating bladder cancer. Although the use of intravesical drugs such as chemical drug and immune revulsive have been the most important part of treating. However these therapeutic methods we use can't control the progression and recurrence of bladder cancer and at the same time the side effects of drugs and drug fast in patients remain the tough problem. For more than twenty years research, wth the development of molecular biology, gene therapy has gradually become mature and is accepting as extremely promising approach to treating cancer after regular treatment such as operation, chemotherapy and radiotherapy. At present an obstacle to the clinical application of gene therapy is a valid target for gene therapy and treatment option, while the discovery of the RNA interference (RNA interference, RNAi) has brought new ideas for gene therapy.RNAi is a conserved mechanism for silencing gene expression in the process of biological evolution phenomenon, it is a small, double-stranded RNA which is stimulated through the degradation of mRNA caused by post-transcriptional gene silencing. In the original organisms,RNAi protect the genome from viruses or other gene fragment inserted, and regulation the expression of many genes during development. The antisense (guide) strand of short double-stranded RNAs is incorporated into an RNA-induced silencing complex that can either suppress protein expression or direct degradation of messenger RNAs that contain homologous sequence. Therefore, RNAi may have an attractive prospects in human genome analysis and tumor treatment. The tumorigenesis and development of tumor has intimate relationship with loss of control in cell cycle regulation. An exception occurs if the cell-cycle control, by the lack of timely repair DNA damage, cells continue to divide, because of genetic instability, can be transformed into cancer cells. The E2F3 transeription factors control expression of genes that are essential for cell proliferation, including key components of both the DNA-replication and cell cycle control machinery. E2F3 is sufficient to induce quiescent cells to enter S phase. Retinoblastoma(Rb) is functionally inactivated in most, if not all, human tumors.The growth-suppressive properties of PRB are thought to be largely dependent upon its ability to regulate the E2F3 transcription factors.Recently Adeno-associated virus vector is researched extensively in gene therapy.because of its broad host,low immunogenicity,high safety and stability,AAVs is considered as one of new generation virus vectors. Hitherto; AAV vector has been used for treating tumor in different animal models, the results show that it is a potential vector.Objective:In this article we have developed Potent RNAi AAVs correponding to E2F3 gene transfected the AAVs into human bladder cancer 5637 cell, assessed the cell cycle and cell apoptosis in bladder cancer 5637 cells.Methods:AAV 293 cells were co-transfected using pAAV-siRNA-E2F3, pRC and pHelper to package by electroporation rAAV-siRNA-E2F3. The cells were under Chloroform-NaCI sediment-Chloroform and ice alcohol to dissociate, purify and concentrate rAAV. Viral particle of purified were assayed by AVSachTM ELISA. Use SDS-PAGE Coomassie brilliant blue staining to observe capsid protein of rAAV. The rAAV-siRNA-E2F3 without endotoxin was extracted successfully and then was transfected into 5637 cells respectively. rAAV-siRNA-E2F3 were transfected into human bladder cancer cell lines 5637 and the transfection efficiency can be acquired by observing green fluorescence of cells. FCM(flow cytometry,FCM) was used to assessed the cell cycle and cell apoptosisb of bladder cancer 5637 cells before and after transfectionin.Results:Under AVSachTM ELISA mensuration, the MOI of rAAV is 5.6×1013 v.p/mL. Under SDS-PAGE and Coomassie brilliant blue staining, we could find three notable viral capsid protein strap of AAV. Green fluorescence can be seen at 30 min and reach to maximum at 48 hours. Detection of cell cycle by flow cytometry showed that:non-treated group and the empty virus group, there are significant differences(P=0.011); non-treated group compared the recombinant plasmid are significant differences (P=0.001); the empty virus group compared the recombinant plasmid are no significant difference (P=0.475). Detection of cell apoptosis by flow cytometry showed that:an empty virus group, apoptosis rate was (7.89±3.75)%; the recombinant plasmid group, apoptosis rate was (24.24±7,76)%.Conclusion:5637 cells can be transfected efficiently by AAV. The rAAV-siRNA-E2F3 was a better transfection reagent for RNAi in 5637 cells.5637 cells can be blocked in G1 phase and induced apoptosis by the recombinant plasmid. Silencing E2F3 may be developed into a potential tool for bladder gene therapy.