Study of construction of siRNA eukaryotic expression vector of Survivin gene and its inhibitory effect on Survivin expression in bladder cancer cells

OBJECTIVE: To construct the specific siRNA (small interference RNA) eukaryotic expression vector of Survivin gene and evaluate its effects on inhibiting Survivin expression and cell apoptosis after transferred into human bladder cancer BIU-87 cells. METHODS: The specific short chain oligonucletide was designed with the help of siRNA software, and the double chain DNA sequences were obtained through annealing after chemosynthesis and inserted into plasmid pSilencer 1.0-U6 to generate the recombinant plasmid pSilencer 1.0-U6-siRNA-Survivin. The recombinant siRNA eukaryotic expression vector was constructed and evaluated by using enzyme digestion analysis and DNA sequencing. After the recombinant eukaryotic expression vector was stably transferred into BIU-87 cells and subjected to G418 selection, and Survivin antisense oligonucleotide (ASODN) was used to compare the inhibitory effect, the growth inhibiting rate (IR)% of BIU-87 cells was observed by MTT test and the apoptosis index (AI) was detected by TUNEL method. The transcription levels of Survivin mRNA and protein expression were respectively detected by semi-quantitive RT-PCR and Western blotting techniques. RESULTS: The results of enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pSilencer 1.0-U6-siRNA-Survivin was successfully constructed. IR and AI, the relative expression of Survivin mRNA and protein of siRNA on BIU-87 cells were significantly higher and lower than those in the groups of ASODN and blank control, respectively (P0.05). CONCLUSIONS: The constructed siRNA eukaryotic expression vector targeting Survivin can effectively down-regulate the transcription and expression of Survivin gene, induce cell apoptosis and inhibit BIU-87 cells growth which provides a new approach and method for the gene therapy of bladder tumor.