耿怀成1 王冰蝉2.RNA 干涉介导的Plk1 沉默对乳腺癌细胞恶性生物表型的影响[J].现代生物医学进展英文版,2011,11(20):3830-3834. |
RNA 干涉介导的Plk1 沉默对乳腺癌细胞恶性生物表型的影响 |
Effect of RNA Interference-Mediated Plk1 Downregulation on MalignantPhenotypes of Breast Cancer Cells |
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DOI: |
中文关键词: Plk1 乳腺癌 RNA 干涉 细胞周期 细胞凋亡 |
英文关键词: Plk1 Breast cancer RNA interference Cell cycle Apoptosis |
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中文摘要: |
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。 |
英文摘要: |
To investigate the effect of Plk1 downregulation on malignant phenotypes of human breast cancer cells. Methods:
Plk1 RNAi vector (pSilencer4.1-shPlk1) 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 Plk1 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/shPlk1 和
MCF-7/shcontrol) were successfully selected. Compared with MCF-7/shPlk1 cells, the levels of Plk1 mRNA and protein expression were
significantly downregulated by 65.8% and 74.4%, respectively (P<0.05). The growth of MCF-7/shPlk1 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/shPlk1 cells was significantly increased by approximately
13.1±2.3% (P<0.05). The expression of active caspase-3 protein in MCF-7/shPlk1 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 Plk1 could specifically downregulate the expression of Plk1 gene. The downregulation of Plk1 could significantly
inhibit growth and in vitro colony formation capacity. Additionally, Plk1 downregulation could also induce cell arrest in G2/M phase of
cell cycle, apoptosis enhancement. Thus, targeting Plk1 will be a potential strategy for the treatment of human breast cancer in future. |
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