| Background and objectivesCancer is caused by the cell proliferation and disequilibrium of apoptosis, and controlled by many extrocellular and intracellular factors. Inhibitor of apoptosis protein (IAP) is a family of intracellular proteins that plays an essential role in the regulation of apoptosis. In many species, the role of IAP is inhibition on apoptosis, and the high IAP expression can cause cell apoptosis deficient, and is closely correlated to tumorigenesis. To search for an effective inhibitor against IAP should take an wonderful perspect for caners' therapy. Recently, Livin was discovered and identified as a novel member of IAP family via homology searches. The distribution of livin is mainly in cancer cells, and no detectable expression in normal adult tissues and elevated levels in placenta, developing tissues, and cancer cell lines. Livin is also correlated to tumorigenesis and drug resistant. So it will be the new target protein in the anti-cancer therapy. In the meanwhile, the RNAi was well known by us, and develops very fast. It can interfere with the genes expression in order to inhibit or kill the tumor cell. Livin was a novel member of IAP family. But whether the esophageal tumor cells could be inhibited by knockdown the Lvin expression through RNAi--it is unknown. So, the experiment plans to construct siRNA vector to aim directly Livin and transfect it into cells of esophageal squamous carcinoma, and then observe the influence about Livin gene on esophageal carcinoma cells growth velocity, cell apoptosis and sensitivity to radioactive rays through regulating the Livin gene expression in the esophageal carcinoma cell lines. MethodsAccording to the principle of designing siRNA target sequence, we used the Takara, Ambion and promega target siRNA sequence design and analysis software to scan human Livin(livinαand livinβ) cDNA exon. 19 bases target siRNA sequence was selected and definited by BLAST homology analysis and a contrast sequence was random assorted. Afterwards, two pairs of DNA oligonucleotides which coded short hairpin RNA sequence were synthesized respectively, and then annealed into double strands DNA. Then these target DNA (double strands DNA) was clone into siRNA express vector (pSUPER-neo) which was cut down by BamHI and ClaI restriction enzyme. The positive clones were selected by BamH I and Cla I restriction enzyme cutting test and sequenced, and then named as pSUPER-neo-Slil,pSUPER-neo-Sli2 and pSUPER-neo-C. Eventually, the siRNA express vector were transfected into human esophageal cancer cell line (EC9706). The siRNA vectors that it can make Livin gene expression in silence and Livinαand Livinβexpression vectors was transfected into esophageal carcinoma cell line EC9706 respectively using packaged by liposome. The Livin mRNA expression was detected by fluorescent quantitation RT-PCR after transfection. The transfected cells growth curve was described and Caspase-3 activity was measured following with the transfected cells apoptosis and sensitivity to radioactive rays detection.Results1. After homologization sequence index and optimization, the target siRNA sequence was ensured, which had 19 bases: GCGTCTGGCCTGCTTCTAT (440-458) and GTCTGGCCTCCTTCTATGA (442-460).2. After the hairpin DNA of siRNA annealing, the results of agar gel electrophoresis analysis showed that there was an obvious stripe whose molecular weight was similar to the design, about 60bp.3. The pSUPER-neo-Sli1,pSUPER-neo-Sli2和pSUPER-neo-C were gotten and identified through BamHI and Cla I restriction enzyme cutting test. 4. After sequenced, the DNA sequences inserted the recombination pSUPER-neo-Sli1 and pSUPER-neo-Sli2 were conformed completely with the design.5. The result of RT-PCR showed that Livin gene expression level were dramatically suppressed.6. The esophageal carcinoma cells transfected into siRNA vector who can make the Livinα/βexpression in silence growth slowed and its apoptosis rate, Caspase-3 activity was increased significantly.Conclusions1. The siRNA oligonucleotide liked hairpin target Livin gene (Livinα,Livinβ) was design.2. The express vector pSUPER-neo-Sli1,pSUPER-neo-Sli2 which aim at Livinαand Livinβwas constructed successfully.3. The express of Livinαor Livinβin esophageal cancer cell EC9706 was obviously decreased after siRNA vectors transfected.4. Regulating Livin gene expression in the esophageal carcinoma cell line EC9706 can change the EC9706 cells biological behaviour effectively. It can be presumed that Livin gene is possible to become a promising molecule target in the tumor gene therapy.
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