对人结肠癌细胞株HT-29转染shRNA重组质粒后Survivin基因变化的研究

目的通过对人结肠癌细胞株HT-29转染shRNA重组质粒后观察Survivin基因的变化。方法设计两条针对Survivin基因CDS区不同位点的siRNA片段并构建shRNA表达载体,重组质粒使用阳离子脂质体法转染结肠癌细胞株HT-29。RT-PCR,Western-blot检测转染后mRNA及蛋白表达。结果转染shRNA重组质粒可有效抑制HT-29细胞Survivin的表达,Survivin mRNA和蛋白表达下调,表明Survivin基因沉默效果较高;两个转染组之间相比pshRNA1组Survivin基因的表达量低于pshRNA2组。结论人结肠癌细胞株中的Survivin基因表达率可通过shRNA重组质粒转染而发生变化;siRNA1和siRNA2序列中Survivin基因表达量存在统计学意义,提示我们在今后有关Survivin基因靶向治疗研究中是否需要考虑到序列的选择以提高作用效果。

Study on the variation of the Survivin gene in human colon cancer cell line HT-29 transfected with shRNA recombinant plasmids

Objective To observe the variation of survivin in human colon cancer cell line HT-29 transfected with shRNA recombinant plasmids.Methods Recombining plasmids to transfect colon cancer cell line HT-29 with cationic liposome by designing 2 siRNA fragments of different sites for survivin gene CDS and constructing its shRNA expression vector.Used RT-PCR,Western-blot to detect the mRNA and protein expression level of the survivin.Results The expression level of the survivin in HT-29 cells was effectively inhibited after transfecting shRNA recombinant plasmids.And the mRNA and protein expression of survivin was down-regulated,which showed that survivin gene was highly silenced.The comparison of the two transfection groups indicated that the expression level of Survivin gene in pshRNA1 group was lower than that of pshRNA2 group.Conclusion Survivin gene expression in human colon cancer cell line HT-29 can be changed through the transfection of its shRNA recombinant plasmids.There exist differences in survivin gene expression between the siRNA1 and siRNA2 sequence prompting us whether it is necessary to consider choosing the sequence to improve the effects in the related Survivin gene targeted therapy in the future.