Li Bin, Sun Baolan, Zhu Jianwei, Zhou Nina, Yang Zhiping, Gu Jianhui
Tumour Biol. 2014 Oct;35(10):10057-66. doi: 10.1007/s13277-014-2312-3.
RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated the expression of RKIP in chronic myelogenous leukemia (CML) K562 cells and the effects of RKIP on the characteristics of K562 cells. The recombinant plasmid pcDNA3.1-RKIP was established and transfected into K562 cells with the help of Lipofectamine 2000. At the same time, the RKIP-siRNA was transfected into K562 cells in another group. The expressions of RKIP in all groups were assayed by Western blot after 48 h. MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to analyze the cell viability. Flow cytometry (FCM) was used to examine the cell cycle and cell apoptosis. Colony forming unit (CFU) assay was used to analyze the effect of RKIP on the clonogenic growth of CML cells. Western blot or luciferase reporter assay was used to detect the effect of RKIP on the level of phospho-ERK1/2 or the transcriptional activity of NF-κB. Western blot analysis showed that the plasmid pcDNA3.1-RKIP or RKIP-siRNA significantly enhanced or decreased RKIP expression (p < 0.01), respectively. In addition, MTT, FCM, and CFU assay indicated that the overexpression of RKIP significantly lowered the cell viability, cell proliferation and the clonogenic growth (p < 0.05), but improved cell apoptosis (p < 0.01). Western blot analysis or luciferase reporter assay showed that the level of phospho-ERK1/2 or the transcriptional activity of NF-κB was strongly inhibited by overexpression of RKIP. All these results could bring us a new perspective for biological therapy in myelogenous leukemia in the future.
RAF激酶抑制蛋白（RKIP）是RAS-丝裂原活化蛋白激酶/细胞外信号调节激酶信号级联反应的负调节因子。我们研究了RKIP在慢性粒细胞白血病（CML）K562细胞中的表达以及RKIP对K562细胞特性的影响。构建了重组质粒pcDNA3.1-RKIP，并借助Lipofectamine 2000将其转染至K562细胞中。同时，将RKIP-siRNA转染至另一组K562细胞中。48小时后，通过蛋白质免疫印迹法检测所有组中RKIP的表达。采用MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）法分析细胞活力。运用流式细胞术（FCM）检测细胞周期和细胞凋亡。采用集落形成单位（CFU）测定法分析RKIP对CML细胞克隆生长的影响。通过蛋白质免疫印迹法或荧光素酶报告基因测定法检测RKIP对磷酸化ERK1/2水平或NF-κB转录活性的影响。蛋白质免疫印迹分析表明，质粒pcDNA3.1-RKIP或RKIP-siRNA分别显著增强或降低了RKIP的表达（p < 0.01）。此外，MTT、FCM和CFU测定表明，RKIP的过表达显著降低了细胞活力、细胞增殖和克隆生长（p < 0.05），但改善了细胞凋亡（p < 0.01）。蛋白质免疫印迹分析或荧光素酶报告基因测定表明，RKIP的过表达强烈抑制了磷酸化ERK1/2水平或NF-κB的转录活性。所有这些结果可能为未来骨髓性白血病的生物治疗带来新的视角。
