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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pSilencer4.1-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/shcontrol)。同MCF-7/shPlk1细胞相比,MCF-7/shPlk1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-7/shPlk1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±2.3%(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。
OBJECTIVE: To investigate the effect of down-regulation of serine/threonine protein kinase Plk1 gene expression on malignant phenotypes in breast cancer cells. METHODS: RNA interference plasmid targeting plk1 gene (pSilencer4.1-shPlk1) was constructed using pSilencer4.1-CMVneo plasmid, respectively. MCF-7 cells were transfected with Lipofectamine 2000 and G418 was used to screen stable transfected cell lines. Semi-quantitative RT-PCR and Western blot were used to detect Plk1 mRNA and protein expression, MTT and colony formation assays were used to detect changes in cell proliferation activity, flow cytometry was used to analyze changes in cell cycle and apoptosis, and finally MCF-7 cells were analyzed for purple. Changes in Chemosensitivity of Suicides (Paclitaxel and Docetaxel). Results: Stably transfected cell lines (MCF-7/shPlk1 and MCF-7/shcontrol) were successfully screened. Compared with MCF-7/shPlk1 cells, the mRNA and protein expression levels of Plk1 in MCF-7/shPlk1 cells were down-regulated by 65.8% and 74.4%, respectively (P<0.05). With MCF-7/shcontrol, the proliferation of MCF-7/shPlk1 cells was significantly inhibited, and the inhibition rate reached 44.9±3.2% on the fifth day (P<0.05). At the same time, the colony forming ability of MCF-7/shPlk1 cells was significantly reduced (P<0.01). Flow cytometry analysis of cell cycle results showed that the proportion of G2/M phase cells in MCF-7/shPlk1 cells increased significantly by 21.1±4.1%, while the proportion of S phase cells decreased significantly (18.5±3.1%; P<0.05). ). Flow cytometry analysis of apoptosis revealed that the apoptosis rate of MCF-7/shPlk1 cells was significantly increased by 13.1±2.3% (P<0.05). At the same time, it was also found that the activated caspase-3 protein in MCF-7/shPlk1 cells increased significantly, Bcl-2 protein decreased significantly, and Bax protein increased significantly. CONCLUSION: The RNA interference vector can specifically down-regulate the expression of Plk1 gene in breast cancer cells, thereby inhibiting the proliferation and in vitro colony formation of breast cancer cells. At the same time, the G2/M phase arrest and the rate of apoptosis of breast cancer cells are significantly increased. . Therefore, biological treatment targeting Plk1 gene is expected to become an important adjuvant treatment strategy for clinical breast cancer in the future.