Experimental Study Of The Interfering Effect On EJ Cell Line Induced By SiRNA Targeting Survivin

Objective To construct eukaryotic expression vectors by using the pSilencer2.0 vector for inhibiting human survivin gene by RNA interference, to detect the effect of the silenced survivin gene on EJ cells. Screen for the recombinant eukaryotic expression vectors which can effective inhibit the expression of survivin gene. To detect it effect on which the generation of EJ cells,cell cycle of EJ cells,apoptosis and apoptosis related genes expression.Methods Two target gene segments were synthesized and cloned into the pSilencer2.0 vector respectively to construct two recombinant eukaryotic expression vectors:pSilencer2.0-SVV1and pSilencer2.0-SVV2 which were identified by enzyme digestion analysis and DNA sequencing. Then the EJ cells were transfected by the recombinant vectors and the interference effect were detected by RT-PCR and Western-blot. The proliferation of the EJ cells was detected by MTT method. The apoptosis index and cell cycle was detected by flow cytometry. The caspase-3,bcl-2 and bax genes expression was detected by RT-PCR.Results (1)Enzyme digestion analysis and DNA sequencing showed that two target segments were exactly cloned into pSilencer2.0 vector.(2)The results of RT-PCR and Western blot indicated that pSilencer2.0-SVV1 and pSilencer2.0-SVV2 vectors could effective knock down the transcription and decrease the expression of survivin mRNA and protein(P<0.05), further more the effect of pSilencer2.0-SVV2 vector was better than the pSilencer2.0-SVV1 vector. (3)After transfected with pSilencer2.0-SVV2 vector, the growth velocity was obviously slow down. The cell numbers which cycle arrest in G2/M phase was significiant increased and the apoptosis index of the EJ cells was increased. The recombinant eukaryotic expression vector (pSilencer2.0- SVV2 vecter) significantly enhanced mRNA of bax(P<0.05),but not those of bcl-2 and caspase-3( P > 0.05).Conclusion The transcription and expression of survivin gene were target inhibited effectively by the recombinant eukaryotic expression vector ( pSilencer2.0-SVV2 vector) in the bladder cancer cell line EJ. After transfected with pSilencer2.0- SVV2 vector, the apoptosis index of the EJ cells was increased and the growth inhibited. It will be worth in the therapy of gene therapy of bladder cancer.