Wei Xiao-Fang, Feng You-Fan, Chen Qiao-Lin, Zhang Qi-Ke
Department of Hematology, Gansu Provincial Hospital, No. 204, Donggang West Road, Lanzhou, 730000 Gansu People's Republic of China.
Cancer Cell Int. 2018 Jul 9;18:96. doi: 10.1186/s12935-018-0587-y. eCollection 2018.
As a disease of hematopoietic stem cell, chronic myeloid leukemia (CML) possesses unique biological and clinical features. However, the biologic mechanism underlying its development remains poorly understood. Thus, the objective of the present study is to discuss the effect of cytidine deaminase (CDA) gene silencing on the apoptosis and proliferation of CML K562 cells.
CDA mRNA expression was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzymatic activity of CDA was measured by a nuclide liquid scintillation method. RT-qPCR and Western blot analysis were used to detect CDA mRNA and protein expression. Cell proliferation, apoptosis and cell cycle were measured by CCK-8 assay and flow cytometry. The expression of proteins relevant to cell proliferation, apoptosis and cell cycle was measured by Western blot analysis. Tumor xenografts were implanted in nude mice to verify the effect of CDA silencing on tumor growth in vivo.
CML and AL patients showed increased mRNA expression and enzymatic activity of CDA. Compared with the blank group, the mRNA and protein expression of CDA in the shRNA-1 and shRNA-2 groups decreased significantly. As a result, the proliferation of K562 cells was inhibited after CDA silencing and the cells were mainly arrested in S and G2 phases, while the apoptosis rate of these cells was increased. In addition, CDA gene silencing in K562 cells led to down-regulated p-ERK1/2, t-AKT, p-AKT and BCL-2 expression and up-regulated expression of P21, Bax, cleaved caspase-3/total caspase-3 and cleaved PARP/total PARP. Finally, CDA gene silencing inhibited tumor growth.
Our study demonstrated that CDA gene silencing could inhibit CML cell proliferation and induce cell apoptosis. Therefore, CDA gene silencing may become an effective target for the treatment of leukemia.
慢性髓性白血病（CML）作为一种造血干细胞疾病，具有独特的生物学和临床特征。然而，其发病的生物学机制仍知之甚少。因此，本研究的目的是探讨胞苷脱氨酶（CDA）基因沉默对CML K562细胞凋亡和增殖的影响。
采用逆转录定量聚合酶链反应（RT-qPCR）检测CDA mRNA表达，用核素液体闪烁法测定CDA的酶活性。RT-qPCR和蛋白质印迹分析用于检测CDA mRNA和蛋白质表达。通过CCK-8法和流式细胞术检测细胞增殖、凋亡和细胞周期。通过蛋白质印迹分析测定与细胞增殖、凋亡和细胞周期相关的蛋白质表达。将肿瘤异种移植物植入裸鼠体内，以验证CDA沉默对体内肿瘤生长的影响。
CML和急性白血病（AL）患者的CDA mRNA表达和酶活性增加。与空白组相比，shRNA-1和shRNA-¬2组中CDA的mRNA和蛋白质表达显著降低。结果，CDA沉默后K562细胞的增殖受到抑制，细胞主要停滞在S期和G2期，而这些细胞的凋亡率增加。此外，K562细胞中的CDA基因沉默导致p-ERK1/2、总AKT、磷酸化AKT和BCL-2表达下调，P21、Bax、裂解的半胱天冬酶-3/总半胱天冬酶-3和裂解的聚（ADP-核糖）聚合酶/总聚（ADP-核糖）聚合酶表达上调。最后，CDA基因沉默抑制了肿瘤生长。
我们的研究表明，CDA基因沉默可抑制CML细胞增殖并诱导细胞凋亡。因此，CDA基因沉默可能成为白血病治疗的有效靶点。
