SHP-1基因在慢性髓性白血病疾病进展中的作用及机制研究  

SHP-1 gene in the disease progression of chronic myeloid leukemia

作　　者：李英华[1] 王兴哲[2] 杨琳[2] 潘玉夏[2] 尚银涛[2] 罗建民[2] 

机构地区：[1] 河北省哈励逊国际和平医院血液科,053000 [2]河北医科大学第二医院血液科,河北省血液病重点实验室,石家庄050000

出　　处：《中华血液学杂志》2014年第12期1074-1078,共5页Chinese Journal of Hematology

基　　金：卫生公益性行业科研专项（201202017）;河北省医学研究课题计划项目（ZD2014090）

摘　　要：目的 探讨SHP-1基因表达与启动子甲基化状态在慢性髓性白血病（CML）疾病进展中的作用及其机制.方法 SYBR Green荧光定量PCR及Western blot方法检测94例CML患者骨髓或外周血单个核细胞中SHP-1基因的表达;甲基化特异性PCR（MSP）方法检测SHP-1基因启动子区CpG岛的甲基化状态;慢病毒表达载体感染K562细胞使SHP-1基因过表达,CCK-8法检测细胞增殖活性,流式细胞术检测细胞的凋亡率及细胞周期分布;荧光探针定量PCR检测BCR-ABL mRNA水平,Western blot方法检测蛋白及磷酸化蛋白的水平.结果 疾病进展期CML（包括加速期和急变期）患者SHP-1 mRNA的相对表达水平为0.79±0.37,较慢性期CML（CP-CML）的1.18±0.64明显降低（P=0.009）,疾病进展期CML患者SHP-1蛋白相对表达水平（0.57±0.02）较CP-CML患者降低（1.02±0.04）（P=0.039）.CP-CML患者SHP-1基因启动子的甲基化阳性率（10/42,23.8％）明显低于疾病进展期CML患者（52/52,100.0％） （P＜0.01）.过表达SHP-1基因的K562-SHP-1细胞与转染空载体的K562-EGFP细胞相比,增殖受抑,凋亡率增加,并出现G0/G1期的细胞阻滞.过表达SHP-1基因降低K562细胞中BCR-ABL蛋白的水平,但不影响BCR-ABL mRNA的转录;过表达SHP-1基因能降低K562细胞MYC蛋白及磷酸化Akt、MAPK、JAK2、STAT5蛋白的表达水平,而对K562细胞中Akt、MAPK、JAK2蛋白总表达水平无影响.结论 SHP-1基因启动子CpG岛甲基化及其基因沉默导致BCR-ABL、Akt、MAPK、JAK2、STAT5、MYC信号通路的异常激活与表达可能与CML疾病进展有关.Objective To investigate the profile of promoter methylation and expression of SHP-1 gene in the progression of chronic myeloid leukemia （CML）.Methods The expression level of SHP-1 mRNA and protein in bone marrow or peripheral blood mononuclear cells from CML patients were detected by Western blot and SYBR Green-based qRT-PCR.The methylation status of SHP-1 were assessed by methylation-specific polymerase chain reaction （MSP） assay.K562 cells were infected with the lentiviral plasmids pEX-SHP-1-puro-Lv105 （K562-SHP-1） or pEX-EGFP-puro-Lv 105 （K562-EGFP）.The levels of proteins and phosphorylated proteins were detected by Western blot.qRT-PCR assay was used to test the level of BCR-ABL mRNA.Results The relative levels of SHP-1 mRNA were sharply decreased in advanced stages CML compared to chronic phase （CP）-CML （0.79±0.37 vs 1.18±0.64,P=0.009）.The level of SHP-1 protein was lower in advanced stages CML compared to CP-CML （0.57±0.02 vs 1.02±0.04,P=0.039）.The frequency of SHP-1 gene promoter methylation at selected loci in CP-CML was 23.8% （10/42）,and the methylated regions were detected in all advanced CML samples （P〈0.01）.SHP-1 was stably transfected into K562 cells and selected with puromycin.Overexpression of SHP-1 inhibited the proliferation and induced the apoptosis of K562 cells,meanwhile leaded to G0/G1 phase arrest.After transfection,the level of BCR-ABL mRNA was not affected in K562-SHP-1 cells （1.32 ± 0.34） compared to K562-EGFP cells （1.18 ± 0.20,P=0.644）,but overexpression of SHP-1 caused a slight decrease in BCR-ABL protein in K562-SHP-1 cells compared to K562-EGFP cells （0.78±0.15 vs 1.27± 0.24,P=0.040）.Overexpression of SHP-1 resulted in a remarkable decrease in MYC protein,phosphorylated forms of JAK2,STAT5,Akt and MAPK.However,the un-phosphorylated forms of these molecules were not significantly affected.Conclusion Decreased expression of SHP-1 caused by aberrant promoter hypermethylation may play a key role in the progression of CML by dysre

关 键 词：白血病 髓系 慢性 BCR-ABL阳性 疾病进展 基因 SHP-1 DNA甲基化 K562细胞 

分 类 号：R733.7[医药卫生—肿瘤]