Cloning of human β-Smac gene and its dubious effect of enhancing Cisplatin-induced apoptosis on gastric cancer cell line SGC7901
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    Abstract:

    Objective:Smac is an established pro-apoptosis protein, which moderates the caspase inbitibion of IAPs. This study will clone humanβ-Smac gene and then investigate whether ectopic overexpression of it would enhance Cisplatin-induced apoptosis on gastric cancer cell line SGC7901. Methods:A pair of PCR primers forβ-Smac gene with lower primer containing a sequence of Flag was designed according to the sequence registered in GeneBank..β-Smac fragements amplificated from SGC7901 genome were cloned into plasmid pcDNA3.1 to construct the desired β-Smac transfectants. The discrepancy inβ-Smac mRNA expression between cells transfected withβ-Smac transfectants or control vectors was investigated by RT-PCR, while the ectopic overexpression of β-Smac protein in cells was detected by western blot. After transfection with either β-Smac transfectants or control vectors for 48h, cells were incubated for another 24 h in the absence or presence of Cisplatin at different concentrations. Then apoptosis was determined by flow cytometry. Results:Nucleotide sequence analysis indicated that the cloned β-Smac sequence was 100% homologous withβ-Smac gene registered in GenBank. Compared with cells transfected with control vectors, the β-Smac mRNA expression was significantly increased and ectopic overexpression of β-Smac protein was only detected in cells transfected with β-Smac transfectants. While the difference of apoptotic activity between cells incubated in 2 μg/ml Cisplatin with and without ectopicβ-Smac overexpression is statistically significant, there are no statistical differences regarding the other three groups. Conclusion:β-Smac transfectant was successfully constructed and ectopically overexpressed in SGC7901. However, the effec tis conditional and probably three is no effect of ectopicβ-Smac overexpression in enhancing Cisplatin-induced apoptosis in SGC7901.

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洪婷婷,陆明洁,葛红梅,束永前,刘 平.β-Smac基因的克隆及过表达对胃癌细胞SGC7901顺铂敏感性的影响[J].南京医科大学学报(自然科学版英文版),2009,29(4):469-474490.

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  • Received:October 31,2008
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