Construction of recombinant adeno-associated virus expressing apoptin and anti-tumor effect on humn bladder cancerin vitro

Objective To construct the recombinant adeno-associated virus (AAV) expressing apoptin and investigate the anti-tumor capabilities of AAV-mediated expression of VP3 to human bladder cancer cells in vitro.Methods In this study the AAV Helper-Free System was used to generate the recombinant adeno-associated virus expressing VP3 gene.The VP3 gene was cloned into the expression vector pAAV-MCS and the recombinant plasmid pAAV-VP3 was confirmed by double enzymatic digestion using EcoR Ⅰ and Sal Ⅰ.The recombinant expression plasmid was co-transfected into the AAV-293 cells with pHelper and pAAV-RC to produce recombinant AAV viral particles.The morphological features of the virus particles were examined under the electron microscopy.The physical titer of recombinant AAV was measured through digoxigenin-labeled CMV probe dot blot method,and the recombinant virus was verified by polymerase chain reaction (PCR) of the exogenous interest genes of apoptin.EJ cells were infected by recombinant virus with 5 × 105 vector genomes per cell.Reverse transcription ( RT)-PCR and Western blotting were performed to detect the transcription and expression of apoptin.Concerning on the anti-tumor activity in vitro,transmission electron microscopy was used to determine the apoptotic morphological changes of EJ cells after rAAV-VP3 infection.The cell cycle and apoptosis of EJ cells were quantified by using flow cytometry at 4th day after infection of rAAV-VP3.Results The expression vector pAAV-VP3 was successfully constructed and the sequence of the VP3 was confirmed by DNA sequencing.The rAAV-VP3 was obtained by three plasmids cotransfection into AAV-293 cells after 72 h.The recombinant adeno-associated virus had a high titer of 5.1 × 1011 v.g./ml,and virus particles could be observed under the electron microscopy.After infection of EJ cells with rAAV-VP3,the transcription and expression of VP3 gene were detected by RT-PCR and Western blotting.The rAAV-VP3-induced morphological changes of apoptosis such as fragmentation and apoptotic body were observed under the transmission electron microcopy.Moreover,the proliferation of EJ cells was slowed down,and the proportion of cells at S phase was decreased and that at G2/M phase was blocked after rAAV-VP3 infection as compared with control group (P ＜0.01 ).Conclusion AAV-mediated VP3 gene transfer may be a new therapeutic technique for the treatment of bladder neoplasms,which may lay a foundation for further studies on anti-tumor effect of bladder cancer in vivo. Key words: VP3 protein;  Bladder neoplasm;  Gene therapy