Lentivirus-mediated p21/Waf1 silencing enhances replicative potential of bladder cancer-specific oncolytic adenovirus and its inhibition effect on proliferation of EJ cells / 肿瘤

ABSTRACT

Objective: To investigate the effects of p21/Waf1 gene silencing on replicative potential of bladder cancer-specific oncolytic adenovirus and the proliferation inhibition of EJ cells. Methods: The shRNA targeting p21/Waf1 gene (p21/Waf1-shNA) was designed and synthesized, then the annealing oligonucleotide fragments were subcloned into pMagic 7.1 vector containing coding gene of green fluorescent protein (GFP) to construct recombinant lentiviral plasmid pLVT1051, which was confirmed by PCR and DNA sequencing. The 293T cells were co-transfected with three plasmids including pLVT1051, pCMV-VSV-G and pCMV-dR8.91 to produce the recombinant lentivirus. The EJ cells were infected with recombinant lentivirus carrying p21/Waf1-shRNA, and the puromycin was used to screen out the stably infected EJ cells. The expression levels of p21/Waf1 mRNA and protein in EJ cells after infection with recombinant lentivirus carrying p21/Waf1-shRNA were detected by real-time fluorescent quantitative-PCR and Western blotting, respectively. The p21/Waf1 gene-silenced EJ cells were infected with bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A, then the expression levels of virus replication-related proteins E1A and Hexon were detected by Western blotting, and the cytotoxic effect on EJ cells was examined by MTT method. Results: Recombinant lentiviral vector carrying p21/Waf1-shRNA targeting p21/Waf1 gene was successfully established and confirmed by DNA sequencing. The recombinant lentivirus was harvested. The stably infected EJ cells were successfully screened out by using puromycin forr two weeks. The expression levels of p21/Waf1 mRNA and protein in EJ cells infected with recombinant lentivirus carrying p21/Waf1-shRNA were significantly reduced (both P < 0.05). The expression levels of E1A and Hexon proteins in p21/Waf1 gene-silenced EJ cells infected with bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A were up-regulated, and the proliferation inhibition of EJ cells was enhanced (P < 0.01). Conclusion: The p21/Waf1 gene-silenced EJ cells are successfully constructed, and p21/Waf1 gene silencing can enhance the replicative ability of bladder cancer-specific oncolytic adenovirus and significantly inhibit the proliferation of EJ cells.