Effect Of Small Interfering RNA Targeting Survivin Gene On Biological Behavior Of Bladder Cancer

Objective 1. To study the influence of small interfering RNA(siRNA) targeting survivin to the signal transduction of bladder cancer cell apoptosis and the anti-apoptotic mechanisms of surviving. 2. To study the influence of siRNA targeting survivin on the biological behavior of bladder cancer. 3. To construct survivin-targeting siRNA-expressing plasmid.Methods 1. One pair of survivin target sequence-specific small interfering RNA(siRNA) was designed and synthesized, then siRNA/liposome complex was used to transfect bladder cancer cell line-T24 with increasing concentrations(50-200nmol/L). MTT assay determined the proliferation of T24 cells. Apoptosis rate were evaluated by flow cytometric analysis. The expression of survivin mRNA and caspase3 mRNA was identified by real-time quantitive PCR. 2. Model of Bladder Carcinoma was found on BLAB/c mice. Mice were randomly divided into 4 groups: (a) untreated mice; (b) mice treated with 5μg siRNA; (c) mice treated with 50μg siRNA; and (d) mice treated with 50μg MMC and the therapeutic efficacy was viewed by measuring the change of volume and pathological change. 3. DNA sequence correspond to siRNA targeting survivin was designed and synthesized ,and cloned into pRNAT-U6.1/Neo to produce plasmid targeting survivin. Order two oligos with cohesive BamHⅠand HindⅢsites. Anneal the two oligos. Cut the vector with BamHⅠand HindⅢ. Ligate the vector with the insert using T4 ligase.The recombinant vector was confirmed by restriction digestion and DNA sequencing.Results 1. SiRNA-survivin efficiently down-regulated surviving expression (mRNA) in a dose and time dependent manner. Its maximum effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down regulated by 75.91﹪.The similar results were found in the inhibition ratio of cell growth,which was 55.29﹪(P<0.05). As revealed by markedly increased caspase3 expression level(mRNA) and apoptotic rate,which were 239.80﹪and 45.70﹪, respectively (P<0.05).2. The mice in the 50μg siRNA group and the MMC groups had a significantly smaller tumor size compared with that observed in the Control group ( P < 0.01). No significant difference in tumor size between the 50μg siRNA and the MMC groups was observed at any time point of this experiment(P =0.548) and no significant difference in tumor size between the 5μg siRNA and the Control groups was observed too(P =0.486) . 3. The PCR product and DNA sequencing showed that the sequence of recombinant vector pRNAT-U6.1/Neo-survivin was successfully constructed, without any base pair mutation.Conclusion 1. The application of siRNA-survivin could markedly inhibit survivin expression in bladder cancer cell line, induce apoptosis and inhibit the growth of the tumor. Maybe it will be a new gene therapy tool for bladder cancer. 2. Maybe survivin inhibits apoptosis through down-regulation of caspase3 expression. 3. The siRNA expressing plasmid pRNAT-U6.1/Neo-survivin was successful constructed. 4.The siRNA expressing plasmid will facilitate the application of RNA interference technique, and lay foundation for further studies of the function of survivin.