Shen Qi, Liu Sichu, Chen Yu, Yang Lijian, Chen Shaohua, Wu Xiuli, Li Bo, Lu Yuhong, Zhu Kanger, Li Yangqiu
Institute of Hematology, Jinan University, Guangzhou 510632, China.
J Hematol Oncol. 2013 Sep 3;6:64. doi: 10.1186/1756-8722-6-64.
Despite the success of imatinib and other tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, and a number of CML patients die due to Abl mutation-related drug resistance and blast crisis. The aim of this study was to evaluate proliferation inhibition and apoptosis induction by down-regulating PPP2R5C gene expression in the imatinib-sensitive and imatinib-resistant CML cell lines K562, K562R (imatinib resistant without an Abl gene mutation), 32D-Bcr-Abl WT (imatinib-sensitive murine CML cell line with a wild type Abl gene) and 32D-Bcr-Abl T315I (imatinib resistant with a T315I Abl gene mutation) and primary cells from CML patients by RNA interference. PPP2R5C siRNAs numbered 799 and 991 were obtained by chemosynthesis. Non-silencing siRNA scrambled control (SC)-treated, mock-transfected, and untreated cells were used as controls. The PPP2R5C mRNA and protein expression levels in treated CML cells were analyzed by quantitative real-time PCR and Western blotting, and in vitro cell proliferation was assayed with the cell counting kit-8 method. The morphology and percentage of apoptosis were revealed by Hoechst 33258 staining and flow cytometry (FCM). The results demonstrated that both siRNAs had the best silencing results after nucleofection in all four cell lines and primary cells. A reduction in PPP2R5C mRNA and protein levels was observed in the treated cells. The proliferation rate of the PPP2R5C-siRNA-treated CML cell lines was significantly decreased at 72 h, and apoptosis was significantly increased. Significantly higher proliferation inhibition and apoptosis induction were found in K562R cells treated with PPP2R5C-siRNA799 than K562 cells. In conclusion, the suppression of PPP2R5C by RNA interference could inhibit proliferation and effectively induce apoptosis in CML cells that were either imatinib sensitive or resistant. Down-regulating PPP2R5C gene expression might be considered as a new therapeutic target strategy for CML, particularly for imatinib-resistant CML.
尽管伊马替尼和其他酪氨酸激酶抑制剂（TKIs）取得了成功，但慢性髓性白血病（CML）在很大程度上仍然无法治愈，许多CML患者因Abl突变相关的耐药性和急变期而死亡。本研究的目的是通过RNA干扰下调PPP2R5C基因表达，评估其对伊马替尼敏感和耐药的CML细胞系K562、K562R（无Abl基因突变的伊马替尼耐药细胞系）、32D-Bcr-Abl WT（具有野生型Abl基因的伊马替尼敏感小鼠CML细胞系）和32D-Bcr-Abl T315I（具有T315I Abl基因突变的伊马替尼耐药细胞系）以及CML患者原代细胞的增殖抑制和凋亡诱导作用。通过化学合成获得编号为799和991的PPP2R5C siRNA。将非沉默siRNA乱序对照（SC）处理、mock转染和未处理的细胞用作对照。通过定量实时PCR和蛋白质印迹分析处理后的CML细胞中PPP2R5C mRNA和蛋白质表达水平，并使用细胞计数试剂盒-8方法测定体外细胞增殖。通过Hoechst 33258染色和流式细胞术（FCM）揭示凋亡的形态和百分比。结果表明，两种siRNA在所有四种细胞系和原代细胞中经核转染后均具有最佳的沉默效果。在处理后的细胞中观察到PPP2R5C mRNA和蛋白质水平降低。PPP2R5C-siRNA处理的CML细胞系在72小时时增殖率显著降低，凋亡显著增加。与K562细胞相比，用PPP2R5C-siRNA799处理的K562R细胞中发现显著更高的增殖抑制和凋亡诱导。总之，RNA干扰抑制PPP2R5C可抑制伊马替尼敏感或耐药的CML细胞增殖并有效诱导凋亡。下调PPP2R5C基因表达可能被视为CML的一种新的治疗靶点策略，特别是对于伊马替尼耐药的CML。
