目的:观察RNA干扰技术能否有效抑制非小细胞肺癌细胞株A549细胞中Polo-like激酶1(Plk1)的表达水平,以及抑制后对A549细胞生长的影响.方法:运用脂质体法,以Plk1为靶点,构建能产生siRNA的质粒载体psiRNA-hH1-Plk1并转入A549细胞.RT-PCR检测Plk1 mRNA表达的变化、Western blotting检测Plk1、cyclin B1、p53蛋白的表达变化、细胞计数分析细胞增殖、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.结果:psiRNA-hH1-Plk1质粒能特异地抑制Plk1基因的表达并使其活性下降,致使cyclin 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期阻滞和凋亡.结论:上述结果提示针对Plkl基因的RNA干扰有望用于肿瘤的基因治疗.%AIM: To investigate whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress Polo- like kinase- 1 (Plk 1 ) expression and its effects in A549 cells. METHODS: A recombinant plasmid containing siRNA targeting Plk1 ( psiRNA - hH1 - Plk1 ) was transfected into A549 cells with Lipofectamine 2000.Expressions of Plk1, cyclin B1 and p53 protein were detected by Western blotting. Cell proliferation was evaluated by direct cell counting, while cell cycle and apoptosis were examined by flow cytometry, and expression of α - tubulin was detected by immunofluorescence. RESULTS: The results demonstrated that sequence specific siRNA targeting Plk1 was capable of suppressing Plk1 expression, and reflecting in lower kinase activity in A549 cells. The level of Plk1 protein was reduced by at least 70％ after 48 h of psiRNA - hH1 - Plk1 treatment relative to controls. Expressions of cyclin B1 and p53 were increased greatly after Plk1 depletion, and cells showed absence of microtubule polymerization and spindle abnormalities in staining for α -tubulin. Growth inhibition, G2/M arrest and apoptosis were observed in psiRNA -hH1 -Plk1 transfected group. CONCLUSION: All these data suggest that siRNA targeted against human Plk1 may be a valuable tool in cancer therapy.
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