Liu Sha, Yuan Fang-Fang, Mi Rui-Hua, Wang Xiao-Jiao, Fan Rui-Hua, Wei Xu-Dong
Department of Hematology, The Cancer Hospital Affiliated to Zhengzhou University & Henan Provincial Cancer Hospital; Zhengzhou 450008, Henan Province, China.
Central Laboratory, The Cancer Hospital Affiliated to Zhengzhou University & Henan Provincial Cancer Hospital; Zhengzhou 450008, Henan Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2017 Apr;25(2):371-376. doi: 10.7534/j.issn.1009-2137.2017.02.011.
To explore the effect of transforming growth factor-β activated kinase-1(TAK1) gene silenced by RNA interference on proliferation inhibition of Kasumi-1 cells induced by AsO and its mechanism.
The experiments were divided into 4 groups, including control group(Kasumi-1 cells treated with non-specific siRNA), TAK1 specific siRNA treated group (Kasumi 1 treated with TAK specific siRNA), AsO treated group (Kasumi 1 cells treated with AsO) and combined treated group (Kasumi 1 cells treated with TAK1 specific siRNA plus AsO). The proliferation inhibition rate of Kasami 1 cells was detected by CCK-8 method, the apoptotic rate of cells was detected by flow eytometry, the expressions of TAK1, phosphorylated c-Jun N-terminal kinase(p-JNK) and apoptosis-related proteins were detected by Western blot.
AsO could inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 20 µmol/L with IC of (3.79±0.36) µmol/L at 24 h, and also inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 10 µmol/L with IC of (2.38±0.17) µmol/L at 48 h, but then the inhibitory effect reached plateau. After treating Kasumi-1 cells with TAK1 siRNA and 3.5 µmol/L AsO for 24 h, the proliferation inhibition rate was (10.86±1.64)% and (49.80±2.19)%, meanwhile the apoptosis rate was (8.47±0.75)% and (24.78±2.14)%, all significantly higher than those in control group (P<0.05, P<0.01). The proliferation inhibition rate and apoptosis rate of the combined treated group were significantly higher than that in control and single treated groups (P<0.05, P<0.01), TAK1 silencing and 3.5 µmol/L of AsO could decrease the expression of TAK1, p-JNK, c-Fos, c-Jun and BCL-2 in different degrees, and increase the expression levels of BAX and the activated (cleaved) caspase-3, 9 with statistically significant differences as compared with control group (P< 0.05). When Kasumi-1 cells were treated with TAK1 specific siRNA plus AsO for 24 h, protein expression levels were all significantly greater than that in the single-treated groups (P< 0.05).
TAK1 silencing and AsO can separately and synergistically inhibit Kasumi-1 cell proliferation which probably relates with the inducing apoptosis via the JNK and mitochondrial pathway. Meanwhile, TAK1 silencing enhances the inhibitory effect of AsO on Kasumi-1 cell proliferation.
探讨RNA干扰沉默转化生长因子-β激活激酶1（TAK1）基因对三氧化二砷（AsO）诱导Kasumi-1细胞增殖抑制的影响及其机制。
实验分为4组，包括对照组（用非特异性小干扰RNA处理的Kasumi-1细胞）、TAK1特异性小干扰RNA处理组（用TAK特异性小干扰RNA处理的Kasumi-1细胞）、AsO处理组（用AsO处理的Kasumi-1细胞）和联合处理组（用TAK1特异性小干扰RNA加AsO处理的Kasumi-1细胞）。采用CCK-8法检测Kasami-1细胞的增殖抑制率，流式细胞术检测细胞凋亡率，蛋白质免疫印迹法检测TAK1、磷酸化c-Jun氨基末端激酶（p-JNK）及凋亡相关蛋白的表达。
AsO在0.5至20 μmol/L之间能以剂量依赖方式抑制Kasumi-1细胞增殖，24 h时半数抑制浓度（IC）为（3.79±0.36）μmol/L；在0.5至10 μmol/L之间48 h时也能以剂量依赖方式抑制Kasumi-1细胞增殖，IC为（2.38±0.17）μmol/L，但随后抑制作用达到平台期。用TAK1小干扰RNA和3.5 μmol/L AsO处理Kasumi-1细胞24 h后，增殖抑制率分别为（10.86±1.64）%和（49.80±2.19）%，同时凋亡率分别为（8.47±0.75）%和（24.78±2.14）%，均显著高于对照组（P<0.05，P<0.01）。联合处理组的增殖抑制率和凋亡率显著高于对照组和单处理组（P<0.05，P<0.01），沉默TAK1及3.5 μmol/L AsO可不同程度降低TAK1、p-JNK、c-Fos、c-Jun和BCL-2的表达，增加BAX及活化（裂解）的半胱天冬酶-3、9的表达水平，与对照组相比差异有统计学意义（P<0.05）。当用TAK1特异性小干扰RNA加AsO处理Kasumi-1细胞24 h时，蛋白表达水平均显著高于单处理组（P<0.05）。
沉默TAK1和AsO可分别及协同抑制Kasumi-1细胞增殖，这可能与通过JNK和线粒体途径诱导细胞凋亡有关。同时，沉默TAK1增强了AsO对Kasumi-1细胞增殖的抑制作用。
