Liu Lin, Zhang Min, Zou Ping, Tian Lei, Liu Fang
Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2006 Apr;14(2):241-6.
The study was purposed to investigate the effect of small interference RNA (siRNA) targeting Polo-like kinase 1 (PLK1) gene on cell cycle progression, proliferation and drug resistance in K562/A02 cells. siRNA plasmid vector specifically targeting PLK1 gene with enhanced green fluorescence protein (EGFP) was transfected into K562/A02 cells. Expressions of PLK1 mRNA and protein were assayed by RT-PCR and Western-blot; cell proliferation was evaluated by direct cell counting after trypan blue staining. Cell cycle and intracellular adriamycin (ADM) accumulation was determined by flow cytometry; 50% inhibition concentration (IC50) of ADM on K562/A02 cells was determined by MTT method. The results showed that, as compared with control cells, siRNA plasmid reduced PLK1 mRNA expression by (34.7 +/- 2.1)% for 24 hours and by (56.6 +/- 1.5)% for 48 hours, PLK1 protein significantly decreased simultaneously by (49.9 +/- 3.2)% and by (62.1 +/- 1.7)%. After being transfected for 24 and 48 hours, the rate of survival cells decreased by 30% and 59% respectively. Forty-eight hours after transfection, the ratio of K562/A02 cells at G2/M increased by 2.77-fold, at the same time, intracellular ADM accumulation increased and the relative efficiency of K562/A02 cells to ADM was 73.8%. It is concluded that PLK1 gene silence can inhibit K562/A02 cell proliferation, induce cell cycle arrest at G2/M, and increase intracellular ADM accumulation, so that enhance cell sensitivity to ADM.
本研究旨在探讨靶向Polo样激酶1（PLK1）基因的小干扰RNA（siRNA）对K562/A02细胞周期进程、增殖及耐药性的影响。将特异性靶向PLK1基因并带有增强型绿色荧光蛋白（EGFP）的siRNA质粒载体转染至K562/A02细胞。采用RT-PCR和Western-blot检测PLK1 mRNA和蛋白的表达；通过台盼蓝染色后直接细胞计数评估细胞增殖。采用流式细胞术检测细胞周期及细胞内阿霉素（ADM）蓄积情况；采用MTT法测定ADM对K562/A02细胞的50%抑制浓度（IC50）。结果显示，与对照细胞相比，siRNA质粒转染24小时后PLK1 mRNA表达降低（34.7±2.1）%，48小时后降低（56.6±1.5）%，同时PLK1蛋白显著降低，分别降低（49.9±3.2）%和（62.1±1.7）%。转染24小时和48小时后，存活细胞率分别降低30%和59%。转染48小时后，K562/A02细胞G2/M期比例增加2.77倍，同时细胞内ADM蓄积增加，K562/A02细胞对ADM的相对敏感性为73.8%。结论：PLK1基因沉默可抑制K562/A02细胞增殖，诱导细胞周期阻滞于G2/M期，并增加细胞内ADM蓄积，从而增强细胞对ADM的敏感性。
