Survivin、Livin共沉默对前列腺癌细胞的联合促凋亡作用

Antitumor effects of silencing Survivin and Livin gene in prostate cancer cell PC3

目的:研究Survivin、Livin基因共沉默对前列腺癌细胞PC-3在细胞周期、细胞增殖、细胞凋亡、对化疗药物敏感性、克隆形成及裸鼠体内成瘤等方面的影响以及Survivin shRNA、Livin shRNA联合促前列腺癌细胞凋亡作用。方法:运用分子克隆技术,构建以Survivin、Livin基因为靶点的、含Survivin启动子的CGM30 miR-30 shRNA载体,慢病毒包装后感染PC-3细胞。免疫印迹实验检测Survivin、Livin的表达变化,MTT、流式细胞术等检测PC-3细胞在细胞凋亡、细胞周期、细胞增殖、对顺铂的敏感性、克隆形成及裸鼠体内成瘤等方面的变化。结果:构建重组慢病毒感染PC-3细胞后,实现了Survivin、Livin基因的共沉默。与单独沉默Survivin或Livin相比,S期PC-3细胞增加了5%-10%。PC-3细胞增殖抑制率和对顺铂的敏感性及凋亡率增加了约20%,克隆形成率明显下降。与阴性对照组比较,裸鼠体内成瘤瘤体重量减小约50%。结论:构建重组慢病毒,成功的在PC-3细胞中实现了Survivin、Livin的共沉默,达到了Survivin shRNA和Livin shRNA二者联合促细胞凋亡的目的。

Objective：To observe the antitumor effects of the tumor specificity RNAi recombinated lentivirus which content the Survivin promoter and Survivin shRNA and Livin shRNA on human prostate cancer cell PC-3. Methods：The CMV promoter of CGM30 miR-30 shRNA vector was replaced by Survivin promoter,then two target gene seg-ments were synthesized and cloned into CGM30 miR-30 shRNA vector respectively. Recombinant vectors was cont-ranfected into 293T cells with pHelper 1. 0 and pHelper 2. 0 plasmid to get packaged recombinant lentivirus. Then PClot. The apoptosis index and cells cycle of PC -3 cells was detected by Flow cytometry. The survival curve of different group PC -3 cells were detected by MTT method. To observe the tumor formation and grown of PC -3 cells which treated by recombinant lentivirus in nudemice xenografts.Results：The results of RT - PCR and Western blot indicated that recombinant lentivirus could knock down the transcription and expression of Survivin and Livin gene only in PC -3 cells. After treated with recombinant lentivirus,the apoptosis index of PC -3 cells was increased about 20%, the number of PC -3 cells of S phase was increase about 5% - 10%,the mean volume and weight of tumors which treated with recombinant lentivirus were less than 50% compared with control groups. Conclusion：Recombinant lentivirus mediated downregulation of Survivin and Livin expression can lead to potent antitumor activity and chemosensitizing effects in prostate cancer not only in vivo but also in vitro,and the antitumor activity and chemosensitizing effects was efficiently then downregulation of Survivin or Livin expression in PC -3 cells unitary.