The Silencing Effects Of Survivin Promoter-Directed SiRNA On Stathmin Gene Transcription In Human HeLa Cells

Objective: To investigate the silencing effects on stathmin gene by survivin gene promoter-driven siRNA vector in HeLa cell line.Methods: (1)Two siRNAs were synthesized according to the stahtmin gene sequence and cloned into the vector pSilencer4.1-CMVneo, which was named pSilencer4.1-S1 vector and pSilencer4.1-S2 vector respectively. Both of them were further identified by restriction endonuclease digestion analysis and DNA sequencing. Then HeLa cells were transfected with pSilencer4.1-S1 and pSilencer4.1-S2, respectively. After G418 selection, the cells were selected and silencing effect were detected by RT-PCR analysis. (2)To construct siRNA eukaryotic expression vector driven by survivin promoter, survivin promoter gene(the sequences was obtained from GeneBank) was reamplified by PCR method and introduced EcoRâ… and BamHâ… restriction enzyme sites by using our previously obtained survivin promoter plasmid as template. The recombinant eukaryotic expression vectors were named pSilencer4.1-surpneo.The recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then HeLa cells were transfected with pSilencer4.1-surp neo and pSilencer4.1- CMVneo as negtive control, then subjected to G418 selection. In G418-resistant cells, the silencing effect was detected by RT-PCR and cell cycle was detected by Flow Cytometry.Results: (1) About 1 kb gene fragment was successfully amplified by PCR method . Sequencing result showed that the sequence of this gene fragment was in according with survivin promoter gene sequence reported in Genbank. (2)Enzyme digestion analysis and DNA sequencing showed that survivin promoter-directed siRNA on stathmin gene were successfully cloned into pSilencer4.1-CMVneo vector. The result of RT-PCR indicated that the transcription of stathmin gene was suppressed by RNAi. Flow Cytometry assay showed that G2/M phase of transfected cells increased significantly, and so the cells were blocked in G2/M phase.Conclusions: (1) The shRNA targeted stathmin gene can effectively knock down stathmin gene expression, which may have potential use in the malignant cancer gene therapy.(2) The transcription of stathmin gene were inhibited effectively by siRNA eukaryotic expression vectors in HeLa cells. All these results show that stathmin gene might be a potential therapeutic target for the treatment of malignant cancers.