Meng Zhen, Wang Dong-Mei, Li Ying-Hua, Liu Xiao, Guo Su-Qing, Luo Jian-Min
Department of Hemotology, Hengshui Harrison International Peace Hospital, Hebei Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Jun;21(3):613-6. doi: 10.7534/j.issn.1009-2137.2013.03.015.
This study was aimed to investigate the expression level of SHP-1 and C-kit genes in acute leukemia HL-60 cells and effect of inhibitor As2O3 demethylation on SHP-1 and C-kit genes expression. RT-PCR was used to detect the expression level of SHP-1 and C-kit mRNA in drug-treated cell group and control group. The methylation specific PCR (MSP) was applied to measure the methylation status of SHP-1 gene in HL-60 cells. The results showed that after being treated with As2O3 the recovery of SHP-1 gene expression was observed in HL-60 cells in which SHP-1 mRNA originally did not expressed, meanwhile the expression level of C-kit mRNA in HL-60 cells with high expression decreased. When HL-60 cells were treated with As2O3 of 1.0, 2.5, 5.0 µmol/L, the demethylation effects was enhanced, the expression of SHP-1 mRNA displayed an ascending tendency, and expression of C-kit mRNA showed an descending tendency in dose-dependent manner (P < 0.05). It is concluded that the absence of SHP-1 mRNA expression in HL-60 cells and recovery of expression after treatment with As2O3 suggest the hypermethylation of SHP-1 gene related with pathogenesis of leukemia, and the abnormal increase of C-kit mRNA expression maybe exist in formation of leukemia. The effect of As2O3 on expression of SHP-1 and C-kit shows dose-dependency, the higher the As2O3 concentration, the higher the SHP-1 expression and the lower the C-kit expression, moreover, the effect of As2O3 shows time-dependency in specific concentration. The SHP-1 mRNA expression negatively relates with C-kit mRNA expression, suggesting that the decrease or absence of SHP-1 expression in leukemia cells weakens the negative regulation on C-kit signaling pathway, thus plays a role in the formation of leukemia.
本研究旨在探讨急性白血病HL-60细胞中SHP-1和C-kit基因的表达水平以及抑制剂三氧化二砷（As2O3）去甲基化对SHP-1和C-kit基因表达的影响。采用逆转录聚合酶链反应（RT-PCR）检测药物处理细胞组和对照组中SHP-1和C-kit mRNA的表达水平。应用甲基化特异性聚合酶链反应（MSP）检测HL-60细胞中SHP-1基因的甲基化状态。结果显示，用As2O3处理后，原本不表达SHP-1 mRNA的HL-60细胞中观察到SHP-1基因表达恢复，同时高表达的HL-60细胞中C-kit mRNA表达水平下降。当HL-60细胞用1.0、2.5、5.0 μmol/L的As2O3处理时，去甲基化作用增强，SHP-1 mRNA表达呈上升趋势，C-kit mRNA表达呈剂量依赖性下降趋势（P<0.05）。结论：HL-60细胞中SHP-1 mRNA表达缺失及As2O3处理后表达恢复提示SHP-1基因高甲基化与白血病发病机制相关，白血病形成过程中可能存在C-kit mRNA表达异常升高。As2O3对SHP-1和C-kit表达的影响呈剂量依赖性，As2O3浓度越高，SHP-1表达越高，C-kit表达越低，此外，As2O3在特定浓度下的作用呈时间依赖性。SHP-1 mRNA表达与C-kit mRNA表达呈负相关，提示白血病细胞中SHP-1表达降低或缺失削弱了对C-kit信号通路的负调控，从而在白血病形成中起作用。
