Zhu Xi-Shan, Lin Zi-Ying, Du Jing, Cao Guang-Xin, Liu Gang
Clinical Research Center, Affiliated Hospital of Guangdong Medical College, Zhanjiang, China E-mail :
Asian Pac J Cancer Prev. 2014;15(12):4773-80. doi: 10.7314/apjcp.2014.15.12.4773.
To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment.
The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment.
The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5 nmol/L~50 nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations (IC50) of siRNA1384, siRNA1276 and siRNA1786 for 24 hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50 nmol/L siRNA transfection.
Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.
研究靶向BCR/ABL mRNA的小干扰RNA（siRNA）对人慢性髓性白血病（CML）K562细胞系增殖和凋亡的影响，为其抗CML治疗的潜在临床应用提供理论依据和实验证据。
从基因库中查找BCR/ABL mRNA的基因序列。根据马克斯 - 普朗克研究所（MPI）和合理的siRNA设计规则选择BCR/ABL mRNA上的靶基因位点，预测候选靶mRNA的二级结构，分析相关热力学参数，并通过BLAST比较靶基因序列以消除任何具有显著同源性的序列。通过MTT法和集落形成抑制试验评估增殖抑制情况。通过流式细胞术（FCM）测定凋亡情况，并通过吉姆萨 - 瑞氏染色鉴定凋亡细胞的形态。使用蛋白质印迹法分析siRNA处理后K562细胞中BCR/ABL融合蛋白的表达。
通过RNA结构软件计算mRNA局部二级结构，并依据生物信息学规则进行特异性siRNA的优化设计。体外设计并合成了5条BCR/ABL siRNA序列。在这5条候选siRNA中，鉴定出3条序列siRNA1384、siRNA1276和siRNA1786对K562细胞生长具有最有效的抑制作用，在12.5 nmol/L至50 nmol/L的siRNA1384作用24、48和72小时后，细胞增殖抑制率接近50%。siRNA1384、siRNA1276和siRNA1786作用24小时的50%抑制浓度（IC50）分别为46.6 nmol/L、59.3 nmol/L和62.6 nmol/L，作用72小时分别为65.668 nmol/L、76.6 nmol/L、74.4 nmol/L。集落形成抑制试验也表明，与对照组相比，siRNA处理组的细胞生长受到抑制。FCM结果显示转染siRNA 24小时后细胞凋亡率增加。膜联蛋白V/PI染色结果表明，用siRNAs（siRNA34、siRNA372、siRNA1384、siRNA1276和siRNA1786）处理后凋亡率增加（1.53%、15.3%、64.5%、57.5%和21.5%）。形态学分析显示，K562细胞暴露于siRNA后出现典型的凋亡形态学变化，如细胞核缩小、碎片化以及“凋亡小体”。蛋白质印迹分析表明，单次转染50 nmol/L siRNA后BCR/ABL蛋白急剧减少。
体外实验中，siRNAs（siRNA1384、siRNA1276和siRNA1786）以浓度依赖性方式显著抑制K562细胞增殖，在50 nmol/L浓度下有效诱导凋亡。K562细胞中的一种抗白血病机制似乎是BCR/ABL靶向蛋白高度下调。siRNAs（siRNA1384、siRNA1276和siRNA1786）可能在CML治疗中具有重要价值。
