目的:探讨沉默转化生长因子β激活激酶(TAK1)基因表达对三氧化二砷(As2O,)抑制人t(8;21)急性髓系白血病细胞株Kasumi-1增殖的影响及其可能机制.方法:实验分为4组:对照组,TAK1特异siRNA转染Kasu-mi-1细胞组,As2 O3作用组及两者联合处理的Kasumi-1细胞组.采用CCK-8法检测细胞增殖抑制率,流式细胞术检测细胞凋亡率,Western blot法检测TAK1、p-JNK及凋亡相关蛋白的表达.结果:在0.5-20 μmol/L范围内,As2O3作用于Kasumi-1细胞24h能够浓度依赖性地抑制Kasumi-1细胞增殖,24 h的IC50值为(3.79±0.36)μmol/L;在0.5-10 μmol/L范围内,As2O3作用于Kasumi-1细胞48 h能够浓度依赖性地抑制Kasumi-1细胞增殖;之后As2O3对Kasumi-1细胞的增殖抑制作用进入平台期,48 h的IC50值为(2.38±0.17)μmol/L.TAK1siRNA转染组和As2O3 3.5 μmol/L作用24 h组,Kasumi-1细胞的增殖抑制率分别为(10.86±1.64)％和(49.80±2.19)％,凋亡率分别为(8.47±0.75)％和(24.78±2.14)％,与对照组相比,差异有统计学意义(P＜0.05);两者联合作用于Kasumi-1细胞的增殖抑制率为(65.63±0.83)％,凋亡率为(68.97±2.94)％,与对照组和各单独组比较,差异均有统计学意义(P＜0.001).TAK1 siRNA转染和As2 O3作用Kasumi-1细胞24 h能不同程度下调TAK1、p-JNK、c-Fos、c-Jun、BCL-2的表达,上调BAX和活化型(cleaved) Caspase-3、9的表达,与对照组比较差异均有统计学意义(P＜0.05),两者联合后该作用进一步加强(P＜0.05).结论:利用RNA干扰技术沉默TAK1表达能够增强As2O3抑制Kasumi-1细胞增殖的作用,其原因可能是通过TAK1下游的JNK信号传导通路,以及线粒体途径诱导细胞凋亡实现的.%Objective:To explore the effect of transforming growth factor-β3 activated kinase-1 (TAK1) gene silenced by RNA interference on proliferation inhibition of Kasumi-1 cells induced by As2O3 and its mechanism.Methods: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),As2O3 treated group (Kasumi 1 cells treated with As2O3) and combined treated group (Kasumi 1 cells treated with TAK1 specific siRNA plus As2O3).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.Results:As2O3 could inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 20 μmol/L with IC50 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 IC50 of(2.38 ± 0.17) μmol/L at 48 h,but then the inhibitory effect reached plateau.After treating Kasumi-1 cells with TAK1siRNA and 3.5 μmol/L As2O3 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 As2O3 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 As2O3 for 24 h,protein expression levels were all significantly greater than that in the single-treated groups (P ＜ 0.05).Conclusion:TAK1 silencing and As2 O3 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 As2O3 on Kasumi-1 cell proliferation.
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