RNA干扰技术抑制Polo-like激酶1表达对A549细胞的影响

Inhibition of Expression of Polo-like Kinase 1 by RNAi Interference in A549 Cells

Polo-like激酶1(Plk1)是参与细胞周期调控的重要分子,已在多种肿瘤中检测到Plk1的高表达,并发现与肿瘤细胞的增殖和预后密切关联.为明确Plk1在肺癌细胞系A549细胞增殖和周期运行中的作用,采用RNA干扰技术,构建能产生siRNA的质粒载体psiRNA-hH1-Plk1并导入A549细胞中.采用RT-PCR检测Plk1mRNA表达的变化,Western印迹检测Plk1、细胞周期蛋白B1、p53蛋白的表达变化,流式细胞术分析细胞周期变化和凋亡;免疫荧光染色检测α微管蛋白的表达.以此观察RNA干扰能否有效抑制Plk1的表达水平,以及抑制后对A549细胞生长的影响.结果表明,psiRNA-hH1-Plk1质粒能特异性地抑制Plk1基因的表达并使其活性下降,细胞周期蛋白B1及p53蛋白的表达水平升高,微管聚集障碍或形成单极的纺锤体,A549细胞增殖减慢,出现G2/M期阻滞并存在细胞凋亡.针对Plk1基因的RNA干扰有望用于肿瘤的基因治疗.

Polo-like kinase 1 （Plk1） has been implicated in various aspects of cell-division cycle and M-phase progression in various eukaryotic organisms. To define the role of Plkl for mitotic progression of lung cancer cells and for neoplastic proliferation, a recombinant plasmid harboring 19-nt-long small interfering （si） RNA （psiRNA-hH1-Plk1） was constructed and tested to selectively downregulate Plkl expression in human lung cancer cell line A549 in vitro transient trasfection with Lipofectamine 2000. The mRNA levels＇of cells transfected with siRNAs were monitored by RT-PCR. Meanwhile, Western blot was used to detect the level of Plkl ,cyclin B1 and p53 protein. Cell proliferation was evaluated by direct cell counting. Cell cycle and apoptosis were examined by flow cytometry, and expression of a-tubulin was detected by immuno-fluorescence. The results showed that sequence specific siRNA targeting Plkl was capable of suppressing Plkl expression, and reflecting in lower kinase activity in A549 cells. The level of Plkl mRNA was efficiently depleted by between 65% and 90% 48 hours after transfection with psiRNA-hH1-Plk1, and reduced Plkl protein level was about 70% after 48 hours relative to controls. Simultaneously, the expression of cyclin B1 and p53 protein were increased greatly after Plkl depletion. Specifically, we stained cells with Hoechst 33258 to monitor the condensation state, and with anti-tubulin antibodies to visualize spindle formation. At 48 hours after transfection, significant proportion of the cells down-expressing Plkl displayed chromosome condensation, absence of microtubule polymerization and abnormalities of spindle assembly, respectivley. In addition, growth inhibition, G2/M arrest and apoptosis were observed in psiRNA-hH1-Plk1 transfected groups. These data indicate that Plk 1, besides playing an important role in regulation of cell cycle progression, is also essential for centrosome mediated microtubule events and consequently for spindle assembly. In addition, siRNA targeted against human Plkl may be valuable tool against a broad spectrum of neoplastic cells.