| Purpose: Bladder cancer ranks frist in incidence among all malignant tumors of the urinary system.Conventional treatments are far from satisfactory for the limited therapeutic efficacy and side-effects.Conditionally replicating adenoviruses(CRADs)are a promising gene therapy method.Therefore,our group constructed a bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1 A,which carries the E1 A gene under the control of the human uroplakin(UPII)promoter and the prostate stem cell antigen enhancer(PSCAE).Ad/PSCAE/UPII/E1 A could selectively replicate in bladder cancer cell lines,thus causing specific tumor cell lysis.In our previous studies,the bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1 A has an excellent specificity only in bladder cancer cells and demonstrated a potent antitumor effect.However,viral replication is attenuated by such low-activity promoters,which compromises viral cytotoxicity.p21/Waf1 is a classical cell cycle-dependent kinase inhibitor however has recently received much attention from researchers because of its inhibitory effects on adenoviral gene therapy.However,the effect of p21/Waf1 on the bladder cancer-specific oncolytic adenovirus is unknown.In this study we constructed a lentiviral vector of p21/Waf1 shRNA,used lentivirus-mediated RNA interference(RNAi)technique to knock down the expression of p21/Waf1 in two bladder cancer cell lines EJ and 5637,established its stable transfected cell lines,so as to pave the way for further research on the effect of p21/Waf1 on the cytotoxic effects of the bladder cancer-specific oncolytic adenovirus,the replicative potential of the adenovirus,the transcriptional activity of the UPII promoter,the apoptosis induced by the adenovirus,and attempted to identify the underlying mechanism.Our results will provide whether this gene has potential use as a therapeutic target for bladder cancer gene therapy.Methods:(1)Construct a lentiviral vector of p21/Waf1 shRNA.One pair of oligonucleotide sequences targeted at human p21/Waf1 mRNA were 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 which was identified byPCR and DNA sequencing.The lentiviral vector and packaging plasmid were co-transfected into293 T cells so as to obtain the lentivirus,and the titer was then determined.EJ and 5637 cells were transfected with recombinant lentivirus and using the best screening concentration of puromycin to get the stable transfection cell lines.RT-PCR and western blot were used respectively to detect the expression of p21/Waf1 after lentivirus infection.(2)The cytotoxic effects of the adenovirus Ad/PSCAE/UPII/E1 A on the p21/Waf1 knockdown bladder cancer cells.MTT cell proliferation assay was used to assess the cytotoxic effects of the adenovirus.(3)The effect of p21/Waf1 on the replicative potential of the adenovirus.The EJ and 5637 cells were infected with Ad/PSCAE/UPII/E1 A and the viral replication assays were performed using Adeno-X Rapid Titer Kit.The expression of the adenovirus replication-related indicators E1 A and hexon were measured.(4)The effect of p21/Waf1 on the transcriptional activity of the UPII promoter.Frist,the mRNA and protein expression of androgen receptor(AR)were measured in the p21/Waf1 shRNA and negative control groups of EJ and 5637 cells.Then the reporter plasmid Rp-PSCAE-UPII-Luc and pCMV-β-gal plasmid were co-transfected into EJ and 5637 cells,and the luciferase activity was detected using the Luciferase Assay System.(5)The apoptosis effects of the adenovirus Ad/PSCAE/UPII/E1 A on the p21/Waf1 knockdown bladder cancer cells.EJ and 5637 cells were infected with Ad/PSCAE/UPII/E1 A,and then the cell apoptosis were detected by flow cytometry.The apoptosis-related m RNA and proteins expression were determined.Results:(1)Recombinant lentiviral vector that expressing p21/Waf1 sh RNA was obtained successfully and we harvested the lentivirus with a titer of 4.12 × 108 Tu/ml.(2)We obtained the stable transfected bladder cancer cell lines,the lentiviral infection rates were all above 90%,lentivirus-mediated shRNA had a significant inhibitory effect on p21/Waf1 expression in EJ and5637 cells.(3)MTT results showed that the cytotoxic effects of Ad/PSCAE/UPII/E1 A was greatly enhanced in the p21/Waf1 knockdown cells of EJ and 5637.(4)The viral replication assays showed an increase production of the viral particles in the p21/Waf1 shRNA group.(5)The levels of both E1 A and hexon were significantly increased in p21/Waf1 knockdown cell lines.(6)Knocking down p21/Waf1 resulted in an increased expression of AR.EJ and 5637 cells that with p21/Waf1 knockdown had a higher expression of luciferase compared with the negative control groups.(7)Ad/PSCAE/UPII/E1 A could induce apoptosis in EJ and 5637 cells,compared with the negative control groups,the adenoviruses induced more cells apoptosis in p21/Waf1 knockdown groups.(8)Ad/PSCAE/UPII/E1 A could induce apoptosis through increasing the expression of Fas,Bax,Caspase 3,Caspase 8,and decreasing the expression of Bcl-2.However,comparing the p21/Waf1 knockdown group to the negative control group,the p21/Waf1 shRNA group displayed increased expression of Fas,Caspase 3,Caspase 8,whereas the expression of Bax and Bcl-2 didnot change significantly.Conclusions:(1)The recombinant lentiviral that expressing p21/Waf1 sh RNA was successfully established and could infect bladder cancer cell lines EJ and 5637 effectively.(2)Lentivirus-mediated sh RNA could efficiently knock down the expression of p21/Waf1 in bladder cancer cell lines.(3)In the p21/Waf1 knockdown cells of EJ and 5637,the cytotoxic effect of Ad/PSCAE/UPII/E1 A was greatly enhanced.(4)Knocking down p21/Waf1 significantly promoted the activity of the UPII promoter.(5)The enhanced cytotoxic effect of Ad/PSCAE/UPII/E1 A in p21/Waf1 knockdown cells was due to the enhanced adenovirus-mediated apoptosis and viral replication. |
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