RNA干涉技术抑制骨肉瘤细胞MG63中Survivin基因表达的研究

Inhibition of Survivin gene expression in human osteosarcoma cell line MG63

目的:构建人Survivin基因的si RNA真核表达载体,探讨其对骨肉瘤细胞MG63中Survivin基因表达的干涉作用。方法:应用pSilencer3.0-H1neo构建Survivin特异性RNA干涉载体,转染MG63细胞,G418筛选稳定转染的细胞。HE染色观察细胞形态学变化,透射电镜观察细胞超微结构。应用RT-PCR、间接免疫荧光和western blot等方法检测Survivin的mRNA和蛋白水平变化。流式细胞仪检测细胞周期变化。结果:成功构建了Survivin基因si RNA真核表达载体Ps-vA和PsvB,获得了稳定转染的MG63细胞。与野生型MG63、阴性对照和MG63/PsvA细胞相比,MG63/PsvB细胞增殖反应明显减弱,差异有统计学意义,P<0.01。MG63/PsvB细胞中Sur-vivin mRNA和蛋白表达减少。与野生型MG63、阴性对照和MG63/PsvA细胞相比,MG63/PsvB凋亡率增加7倍(P<0.01)。结论:特异性si RNA能够明显抑制Survivin基因在MG63细胞中的表达,为进一步研究survivn在MG63细胞中的生物学功能和作用机制奠定了基础。

OBJECTIVE：To explore the blocking effect of siRNA on the expression of Survivin in gene in osteosarcoma cell line MG63. METHODS： According to Survivin eDNA coding sequence, the specific RNA interference （RNAi） fragments targeting Survivin gene were designed and synthesized, which were cloned into pSilencer 3.0-H1 neo plasmid vector, and the shRNA eukaryotic expression vector siRNA Survivin targeting Survivin gene was constructed. After the vector was constructed, MG63 cells were transfected with negative control vector siRNA neg or RNAi vectors and selected by G418. Expression of mRNA and protein of Survivin in the stable transfected cells was investigated seperately by RT-PCR, Western blot, immunofluorescence microscopy, and flow cytometry. RESULTS： The specific siRNA eukaryotic expression vector PsvA and PsvB targeting Survivin gene were constructed successfully. The stable transfectants containing negative control vector siRNA neg, PsvA and PsvB were obtained. Expression of mRNA and protein of Survivin was inhibited significantly in MG63/PsvB cells. Whereas Survivin gene expression levels were hardly changed in the other groups. Otherwise, MG63/PsvB had significant decreases in cell number compared with other transfectants. Analysis of DNA content in PsvB transfectants revealed a 7-fold increase in the fraction of sub-G1 peak （apoptotic peak） as compared with vector control and PsvA transfectants, under the same experimental conditions,P〈0.01. CONCUSlON：Survivin gene expression can be suppressed markedly by specific shRNA in MG63 cells, which establishes the experimental foundation for further studying the biological functions and its mechanisms of Survivin in MG63 cells.