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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.
Polym-like kinase 1 (Plk1) is an important molecule involved in cell cycle regulation and has been detected in a variety of tumors Plk1 high expression and found to be closely related to the proliferation and prognosis of tumor cells.To clarify the Plk1 in lung cancer cell line A549 RNA interference technique was used to construct plasmid vector psiRNA-hH1-Plk1 that can generate siRNA and then introduced into A549 cells.The changes of Plk1 mRNA expression were detected by RT-PCR and the expression of Plk1, cyclin B1 and p53 were detected by flow cytometry, the cell cycle and apoptosis were analyzed by flow cytometry, and the expression of α-tubulin was detected by immunofluorescence staining.It was observed whether RNAi could effectively inhibit the expression of Plk1, The results showed that psiRNA-hH1-Plk1 plasmid could specifically inhibit the expression of Plk1 gene and decrease its activity, the expression of cyclin B1 and p53 protein increased, and the microtubule aggregation disorder or monopolar Of A549 cells, the proliferation of A549 cells slowed down, G2 / M phase arrest and cell apoptosis appeared.The RNAi targeting Plk1 gene was expected to be used in the gene therapy of tumors.