Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells
Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells
复制标题
DOI：
10.1093/jnci/94.24.1863
复制
发表时间：
2002-12-18
影响因子：
10.3
通讯作者：
Strebhardt, K
中科院分区：
文献类型：
--
作者：
Spänkuch-Schmitt, B;Bereiter-Hahn, A;Strebhardt, K
背景：polo 样激酶 1 (PLK1) 在有丝分裂进展中具有多种功能，在多种人类肿瘤中表达升高。为了研究 PLK1 在肿瘤增殖中的作用，我们使用了 RNA 干扰技术。方法：用针对人 PLK1 或核纤层蛋白基因的小干扰 (si) RNA 转染来自几种不同癌细胞系（MCF-7 乳腺癌细胞、HeLa S3 宫颈癌细胞、SW-480 结肠癌细胞和 A549 肺癌细胞）的细胞。 Northern 和蛋白质印迹分析用于检查转染的癌细胞和正常细胞（人乳腺上皮细胞 [HMEC]）中的 PLK1 基因表达。还通过荧光显微镜和荧光激活细胞分选分析监测转染 siRNA 的细胞的表型、增殖和细胞周期分布。结果：所有转染低剂量针对 PLK1 的 siRNA 的癌细胞系的 PLK1 mRNA 和蛋白质水平均大大降低。抑制作用最强的siRNA4，转染24小时后，MCF-7细胞中PLK1 mRNA减少70%，MCF-7细胞中PLK1蛋白减少95%。转染后48小时，细胞增殖减少66%至99%，转染细胞凋亡从1%-5%增加至13%-50%。转染的 SW-480 细胞有丝分裂被抑制，它们的中心体失去了使微管成核的能力。 HMEC 吸收 siRNA 的效率低于癌细胞，并且用针对 PLK1 的 siRNA 转染不会抑制其增殖。结论：PLK1 功能似乎对于中心体介导的微管事件以及纺锤体组装至关重要。针对人类 PLK1 的 siRNA 可能是作为抗增殖剂的有价值的工具，其在极低剂量下表现出针对广谱肿瘤细胞的活性。
Background: Expression of polo-like kinase-1 (PLK1), which has several functions in mitotic progression, is elevated in a broad range of human tumors. To investigate the role of PLK1 in neoplastic proliferation, we used the technique of RNA interference. Methods: Cells from several different cancer cell lines (MCF-7 breast cancer cells, HeLa S3 cervical cancer cells, SW-480 colon cancer cells, and A549 lung cancer cells) were transfected with small interfering (si) RNAs targeted against the human PLK1 or lamin genes. Northern and western blot analyses were used to examine PLK1 gene expression in transfected cancer cells and normal cells (human mammary epithelial cells [HMECs]). The phenotype, proliferation, and cell cycle distribution of cells transfected with siRNAs were also monitored by fluorescence microscopy and fluorescence-activated cell sorting analysis. Results: All cancer cell lines transfected with low doses of siRNAs targeted to PLK1 had greatly decreased levels of PLK1 mRNA and protein. siRNA4, which had the strongest inhibitory effect, reduced PLK1 mRNA in MCF-7 cells by 70% and PLK1 protein in MCF-7 cells by 95% 24 hours after transfection. Cell proliferation was reduced by between 66% and 99% 48 hours after transfection, and apoptosis was increased from 1%-5% to 13%-50% in transfected cells. Transfected SW-480 cells were mitotically arrested, and their centrosomes had lost the ability to nucleate microtubules. HMECs took up siRNAs less efficiently than cancer cells, and transfection with siRNAs targeted to PLK1 did,not inhibit their proliferation. Conclusions: PLK1 function appears to be essential for centrosome-mediated microtubule events and, consequently, for spindle assembly. siRNAs targeted against human PLK1 may be valuable tools as antiproliferative agents that display activity against a broad spectrum of neoplastic cells at very low doses.