RNA干涉介导的Plk1沉默对乳腺癌细胞恶性生物表型的影响

目的：研究乳腺癌细胞中丝／苏氨酸蛋白激酶Plk1基因表达下调后对其恶性生物表型的影响。方法：利用pSitencer4．1-CMVneo质粒，分别构建针对Plk1基因的RNA干涉载体（pSilencer4．1-shPlk1），利用脂质体Lipofectamine2000转染MCF-7细胞，G418筛选稳定的转染细胞系。半定量RT—PCR和Western blot分别检测Plk1基因mRNA和蛋白表达，MTT和克隆形成试验检测细胞增殖活性的变化，流式细胞仪分析细胞周期和凋亡的变化，最后分析MCF-7细胞对紫杉类药物（紫杉醇和多西他赛）化疗敏感性的变化。结果：成功筛选了稳定转染细胞系（MCF-7／shPlk1和MCF-7／shcontro1）。同MCF-7／shPlk1细胞相比，MCF-7／shPtkl细胞中Plk1基因mRNA和蛋白表达水平分别下调65．8％和74．4％（P〈0．05）。同MCF-7／shcontrol，MCF-7tshPlk1细胞增殖速度显著抑制，到第5天时抑制率达到44．9±3．2％（P〈0．05）。同时，MCF-7／shPlk1细胞的克隆形成能力显著降低（P〈0．01）流式细胞仪技术分析细胞周期结果表明：MCF-7／shPlk1细胞的G2／M期细胞比例显著增加了21．1±4．1％，而S期细胞比例则显著降低了（18．5±3．1％；P〈0．05）。流式细胞仪技术分析细胞凋亡结果表明：MCF-7／shPlk1细胞的凋亡率约显著增加了13．1±213％（P〈0．05），同时还发现：MCF-7／shPlk1细胞中激活的caspase-3蛋白显著增加，Bcl-2蛋白显著降低，而Bax蛋白则显著增加。结论：RNA干涉载体能特异性下调乳腺癌细胞中Plk1基因的表达，从而抑制乳腺癌细胞的增殖和体外克隆形成能力，同时诱导乳腺癌细胞的G2／M期阻滞和细胞凋亡率显著增加。因此，靶向Plk1基因的生物治疗有望成为未来临床乳腺癌的一个重要的辅助治疗策略.

Effect of RNA Interference-Mediated Plkl Downregulation on Malignant Phenotypes of Breast Cancer Cells

To investigate the effect of Plkl downregulation on malignant phenotypes of human breast cancer cells. Methods： Plkl RNAi vector （pSilencer4.1-shPlkl） was constructed and stably transfected into breast cancer cell line （MCF-7） by using Lipofectamine 2000 agent. Semi-quantitative RT-PCR and Western blot assays were performed to detect the expression of Plkl mRNA and protein in the stably transfected MCF-7 cells. MTT and colony formation assays were performed to detect the cell viability. Flow cytometry assay was performed to detect cell cycle and apoptosis. Results： The stably transfected MCF-7 cells （MCF-7/shPIk1和 MCF-7/shcontrol） were successfully selected. Compared with MCF-7/shPlkl cells, the levels ofPlkl mRNA and protein expression were significantly downregulated by 65.8% and 74.4%, respectively （P〈0.05）. The growth of MCF-7/shPIkl was significantly inhibited, and the highest inhibitory rate was 44.9＋ 3.2% at day 5 （P〈0.05）. Flow cytometric analysis of cell cycle showed that the rate of G2/M phase cells was significantly increased by 21.1 ±4.1% and the rate of S phase cells was significantly decreased by 18.5±3.1% （P〈0.05）. Flow cytometric analysis of apoptosis showed that the apoptotic rate of MCF-7/shPlkl cells was significantly increased by approximately 13.1± 2.3% （P〈0.05）. The expression of active caspase-3 protein in MCF-7/shPlkl cells was significantly increased. Moreover, the expression of Bcl-2 protein was significantly downregulated, but the expression of Bax protein was significantly upregulated. Conclusion： RNAi vector targeting Plk 1 could specifically downregnlate the expression of Plk 1 gene. The downregnlation of Plk 1 could significantly inhibit growth and in vitro colony formation capacity. Additionally, Plkl downregnlation could also induce cell arrest in G2/M phase of cell cycle, apoptosis enhancement. Thus, targeting Plkl will be a potential strategy for the treatment of human breast cancer in future.