Silencing Of E2F3 Expression In Bladder Cancer Cell Line 5637 Using RNAi With AAV Virus Expressing Vector

Bladder cancer is one of the most common urological malignant diseases,most of bladder cancer is transitional-cell carcinoma.The most important features of urothelial cancers of the bladder are metachronous or synchronous multifocal occurrence with high frequeney.Operation has been the main measure in treating bladder cancer,but after operation bladder cancer easily recurs and the recurrence cancer often increases its progression.For this reason the use of intravesical drugs such as chemieal drug and immune revulsive have been the most important part of treating.However these therapeutie methods we used cannot control the Progression and recurrence of bladder cancer and at the same time the side effects of drugs and drug resistence in Patients remain the tough problem.For more than twenty years research 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.The discovery that RNAi works in mammlian cells has sparked intense investigation into its role in normal mammlian cell function,its use as a tool to understand or screen for genes functioning in cellular pathways in healthy and diseased cells and animals,and its potential for therapeutic gene silencing.RNA interference(RNAi) is an evolutionary conserved mechanism for silencing gene expression.In primitive organisms,RNAi Protects the genome from viruses and other insertable genetic elements and regulates gene expression 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(s).RNAi may provide an important new therapeutic scheme for treating illnesses especially for cancer.On the condition of high safety,efficiency vector,RNAi may process in cells favorably 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.The E2F 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. The overexpression of E2F3 in human bladder cancer cells is a common phenomenon, now many scholar think that The tumorigenesis and development of tumor has intimate relationship with the overexpression of E2F3,knock-down of the expression of E2F3 may restrain development of bladder cancer.Objective:In this article we want to know if AAV can be used effectively in RNAi of Bladder cancer cell line and wheher it can take siRNA into cells.To answer these questions we did the following jobs:we have developed effective RNAi AAVs correponding to E2F3 gene transfected the AAVs into human bladder cancer cell line 5637,assessed the E2F3 mRNA and protein levels in 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-NaCl 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.RT-PCT was used to determine if rAAV-siRNA-E2F3 can downregulate the expression of E2F3 mRNA.The silencing effect can also be detected by Western blot in protein levels.Results:Under AVSachTM ELISA mensuration,the MOI of rAAV is 5.6×10¹³v.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. Relative to control,rAAV-siRNA-E2F3 induced 69.84%(P＜0.01) inhibition of E2f3 mRNA treatment in 5637 cells.Comparing with control,E2F3 protein levels of 5637 cell significantly reduced and inhibition rate is 83.27%(P＜0.01)Conelusion:5637 cells can be transfected efficiently by AAV.The rAAV-siRNA-E2F3 was a better transfection reagent for RNAi in 5637 cells.The sequence specific siRNA showed a block effect in downregulation of E2F3 gene expression,Silencing E2F3 may be developed into a potential tool for bladder gene therapy.