三氧化二砷抑制NB4细胞增殖机制的探讨

Study on the mechanism of arsenic trioxide inhibiting NB4 cells proliferation

目的通过检测NB4细胞活化JAK1蛋白水平探讨三氧化二砷（As2O3）对其增殖抑制的分子生物学机制。方法利用Westernblot检测NB4细胞JAKl蛋白表达及磷酸化水平；用小分子RNA（siRNA）干扰NB4细胞JAKl蛋白的表达及转染JAK1质粒使NB4细胞内JAK1过表达；应用MTT法及锥虫蓝拒染法观察在JAK1基因沉默或过表达的状态下，As2O3抑制NB4细胞增殖的变化情况；在此基础上利用Westernblot方法进一步检测磷酸化JAK1蛋白（p-JAK1）及细胞周期抑制蛋白P21的变化。结果NB4细胞内可检测到JAKJ蛋白稳定表达，但未检测到其磷酸化；As2O3作用NB4细胞后，JAK1磷酸化水平增强；JAK1siRNA转染NIM细胞后JAK1蛋白表达水平明显低于非特异性siRNA转染组（即laminsiRNA转染组）及空白对照组；且在JAK1siRNA转染组As2O3抑制NIM细胞增殖作用减弱，4μmol／LAs2O3对JAK1siRNA转染组NB4细胞增殖抑制率为49．12％，较非特异性siRNA组（74．58％）及空白对照组（72．33％）明显下降；JAKl质粒转染NB4细胞后野生型及突变型JAK1质粒组较空质粒组JAK1蛋白表达水平显著升高，且在野生型JAK1质粒转染组，As2O3抑制NB4细胞增殖作用增强，4μmol／L As2O3对野生型JAK1质粒转染组NIM细胞增殖抑制率为69．53％，较突变型JAKl质粒组（37．26％）及空质粒组（39．61％）明显升高；As2O3作用NIM细胞后P21蛋白的表达水平上调。结论JAK1蛋白在NB4细胞内稳定表达但没有活性；As2O3通过激活JAK1蛋白抑制NB4细胞增殖；As2O3激活JAK1蛋白后通过上调P21的表达而抑制NB4细胞增殖。

Objective To explore the molecular mechanisms of arsenic trioxide （As2O3 ） inhibiting NB4 cells proliferation. Methods The Janus kinase 1 （JAK1） protein level and its phosphorylation level in NIM cells was detected by Western blots. NB4 cells were transfected with JAKI siRNA or JAK1 plasmid to make JAK1 gene silenced or overexpressed. The inhibition of NB4 cells proliferation was measured by MTT assay and Trypan blue exclusion respectively. The variation of phosphorylatioa level of JAK1 and the cell cycle inhibitor P21 were determined by Western blots. Results JAKl protein was expressed stably in NB4 cells, with no phosphorylation. The phosphorylation of JAK1 was enhanced after the NB4 cells treated with As2 03. After NB4 cells transfected with JAK1 siRNA, the expression level of JAKI was obviously lower than that of in the non-specific siRNA group and blank control group. The effect of As2O3 inhibiting NB4 cells proliferation was weaker in the JAK1 siRNA transfeeted group. The inhibiting rate of 4 μmol/L As2O3 on NB4 cells proliferition of JAK1 siRNA group was 49.12% being lower than that of the non-specific siRNA group （74.58%） and control group （72.33%）. After NB4 cells transfeeted with JAK1 plasmid, the JAK1 expression level in wild-type and mutant type plasmid groups were significantly higher than those in the empty plasmid group, moreover the effect of As203 inhibiting proliferation was stronger in wild-type plasmid group. The inhibiting rate of 4 μmol/L As2O3 on NB4 cells proliferition of wild-type plasmid group was 69.53% being higher than that of the mutant eype JAK1 plasmid group （37.26%） and the empty plasmid group （39.61%）. The expression level of P21 was up-regulated after the NB4 cells treated with As203. Conclusion JAK1 is expressed stably in NB4 cells, but has no activity. Arsenic trioxide inhibits the proliferation of NB4 cells through activating the JAK1. P21 is up-regulated after arsenic trioxide activated the JAK1 to inhibit the proliferation of NB4 cells.