K562细胞中DNMT1与SHP-1启动子2甲基化关系的研究  

Relationship between DNMTl and Methylation of SHP_1 Promoter 2 in K562 Cells

作　　者：刘学东 刘晓[2] 郭秀芬[2] 罗建民[3] 李英华[2] LIU Xue-Dong1 , LIU Xiao2, GUO Xiu-Fen2, LUO Jian-Min3 , LI Ying-Hua2(1Hengshui Blood Center; 2 Department of Hematology, Harrison International Peace Hospital, Hengshui 053000, Hebei Province, China; 3 Department of Hematology, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, Hebei Province, Chin)

机构地区：[1]衡水市中心血站 [2]哈励逊国际和平医院血液科,河北衡水053000 [3]河北医科大学第二医院血液科,河北石家庄050000

出　　处：《中国实验血液学杂志》2018年第2期401-406,共6页Journal of Experimental Hematology

基　　金：河北省医学研究课题计划项目ZD2014090

摘　　要：目的:探讨慢性髓系白血病(CML)急变细胞系K562细胞中DNA甲基转移酶1(DNMT1)与造血细胞磷酸酶(SHP-1)基因表达及其启动子2的甲基化状态的关系。方法:分析NCBI数据库中SHP-1基因启动子2启动子区序列。将携带DNMT1sh RNA的psi HIV-m U6-sh DNMT1和樱桃色荧光蛋白(mcherry FP)psi HIV-m U6-control的慢病毒干扰载体感染K562细胞系。应用甲基化特异的聚合酶链反应(MSP)和亚硫酸氢盐修饰后测序法(BSP)检测K562细胞中SHP-1基因启动子2的甲基化状态,Western blot检测SHP-1、DNMT1蛋白的表达水平,SYBR Green荧光定量PCR检测SHP-1 m RNA的表达。结果:SHP-1基因启动子2在-353 bp-+182 bp区有22个Cp G岛,K562细胞中SHP-1基因启动子2异常高甲基化且SHP-1蛋白表达缺失。K562细胞转染psi HIV-m U6-sh DNM T1后DNM T1表达水平为0.48±0.06,较转染psi HIV-m U6-control组明显降低(1.33±0.19)(t=4.18,P=0.014)。K562细胞中SHP-1蛋白表达缺失,转染psi HIV-m U6-sh DNM T1后K562细胞SHP-1蛋白恢复表达,转染psi HIV-m U6-sh DNM T1的K562细胞中SHP-1 m RNA相对表达水平(14.23±3.83)较转染psi HIV-m U6-control组(1.031±0.156)高(P=0.004)。DNMT1基因沉默后SHP-1基因启动子2中Cp G岛去甲基化。结论:K562细胞中DNMT1与SHP-1启动子2的异常甲基化及沉默相关。Objective： To investigate the relationship of DNA methyltransferase 1 （ DNMT1 ） with hematopoietic cell phosphatase （ SHP-1 ） gene expression and promoter 2 methylation status in cell line K562. Methods ： The promoter sequence of SHP-1 gene promoter 2 in NCBI database was analyzed, the K562 cells were transfected with the lentiviral plasmids--the specified retroviral vector psiHIV-mU6-shDNMT1 and psiHIV-mU6-mcherryFP-control. The methylation status of SHP-1 gene promoter 2 in K562 cells was detected by methylation - specific polymerase chain reaction （MSP） and bisulfite-modified sequencing （BSP）. Western blot was used to detect the protein expression level of SHP-1 and DNMT1, the SYBR Green fluorescence quantitative PCR was used to detect the expression of SHP-1 mRNA. Results： It was found that the promoter 2 of SHP-1 gene located between - 577 bp to ＋ 300 bp, and 22 CpG sites contained between - 353 bp - ＋ 182 bp were aberrantly hyperrnethylated and the SHP-1 could not be detected in K562 cells. In vitro, the detection demonstrated that the expression level of DNMT1 in K562 cells transfected with psiHIV-mU6- shDNMT1 was 0.48±0. 06 significantly lower than that of psiHIV-mU6-control group （ 1.33±0.19） （ t = 4.18, P 〈 0.05 ）. The expression of SHP -1 mRNA in K562 cells transfected with psiHIV-mU6-shDNMT1 was significantly higher than that in K562 cells transfected with psiHIV-mU6-shDNMT1 （ 14.23±3.83 vs 1. 031 ±0. 156 ） （ P 〈 0.01 ）. DNMT1 silencing induced demethylation of the 22 CpG sites located in the SHP-1 promoter 2, and SHP-1 gene was re- expression in K562 cells. Conclusion： The DNMT1 in K562 cells relates with the hypermethylation and silencing of SHP-1 promoter in K562 cells.

关 键 词：SHP-1 DNMT1 K562细胞 启动子甲基化 

分 类 号：R733.72[医药卫生—肿瘤]