靶向生存素基因siRNA诱导骨肉瘤细胞株U-2OS的凋亡

Survivin gene-targeted siRNA inducing apoptosis of human osteosarcoma cell line U-2OS

目的:体外转录合成生存素(survivin)基因siRNA,观察其在骨肉瘤细胞株U-2OS中抑制survivin基因后细胞增殖及细胞凋亡情况。方法:将U-2OS细胞分为A组(空白对照组)、B组(非特异性转染组)、C组(特异性转染组),在荧光显微镜下观察转染前后细胞形态的改变,用RT-PCR和Western blot法分别检测转染前后survivin基因mRNA和蛋白的干涉效果,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡情况,并比较各组结果。结果:荧光显微镜下可见转染后细胞增殖变慢, 凋亡细胞增加;RT-PCR结果显示survivin siRNA明显抑制survivin基因mRNA表达。Western blot法检测C组蛋白阳性表达率比A、B两组显著降低(P<0．01);特异性转染组的细胞增殖缓慢,与其它两组比较有显著性差异(P<0.01)。流式细胞仪检测C组细胞凋亡率与其他两组比较有显著性差异(P<0．01)。结论:体外转录合成特异性survivin siRNA能有效抑制U- 2OS细胞中survivin,in mRNA及蛋白表达,从而抑制细胞增殖,诱导细胞凋亡。RNA干扰技术为骨肉瘤的基因治疗提供了一种新策略。

Objective ： To investigate the proliferation and apoptosis of human osteosarcoma cell line U-2OS after survivin gene was knocked down by siRNA synthesized in vitro. Methods： U-2OS cells were divided into 3 groups： Group A （blank control） , group B （transfected with non-specific siRNA） and group C（ transfected with survivin-specific siRNA）. The morphological changes of U-2OS cells were observed with fluorescent microscope. The expression of survivin mRNA and protein were detected by reverse transcription polymerase chain reaction （RT-PCR） and Western blotting. The antiproliferative effects were assessed by MTT and the rate of apoptosis was examined by flow cytometry in 3 groups. The results of the 3 groups were analyzed and compared. Results： Survivin specific siRNA significantly down-regulated mRNA and protein expression of survivin. Expression of survivin protein in group C was significantly lower than those in group A and B （ P 〈 0.01 ） ; Group C also showed a slower proliferation than the other 2 groups （ P 〈 0.01 ）. The apoptosis rate in group C was significantly higher than those in other 2 groups （P 〈 0.01 ）. Conclusion： siRNA can efficiently suppress survivin gene expression, inhibit proliferation and induce apoptosis in osteosarcoma cell line U-2OS.