巢式MSP检测砷剂诱导人多发性骨髓瘤U266细胞系p16基因去甲基化及转录  

n-MSP Detection of p16 Gene Demethylation and Transcription in Human Multiple Myeloma U266 Cell Line Induced by Arsenic Trioxide

作　　者：傅海英[1] 沈建箴[1] 沈松菲[1] 周华蓉[1] 

机构地区：[1]福建医科大学附属协和医院血液科,福建省血液病研究所,福州350001

出　　处：《中国实验血液学杂志》2007年第1期79-85,共7页Journal of Experimental Hematology

基　　金：福建省百千万人才工程基金资助项目;编号303052801

摘　　要：本研究探讨用高灵敏度的DNA甲基化检测方法及DNA克隆测序分析法检测三氧化二砷(As2O3)的去甲基化作用,并对其可能的去甲基化作用机制进行分析。采用巢式甲基特异性PCR法(nested-methylation specificPCR,n-MSP)、DNA克隆测序分析法检测As2O3作用前后U266细胞株p16基因甲基化状态,应用RT-PCR检测p16、DNA甲基转移酶1(DNMT1)、DNMT3A、DNMT3B基因mRNA的表达,以生长曲线、MTT法、集落形成实验检测As2O3对骨髓瘤细胞生长和增殖的抑制作用。利用流式细胞仪DNA含量分析法探讨As2O3对多发性骨髓瘤细胞系U266周期的影响。结果表明:①未处理组U266细胞基因组DNA的胞嘧啶保持不变,而经As2O3作用的U266细胞基因组DNA的胞嘧啶均已变为胸腺嘧啶,这说明U266细胞存在p16基因甲基化,As2O3作用后p16基因异常甲基化的现象被逆转;②未处理组细胞p16基因不表达,As2O3作用72小时后p16基因表达增强,0.5μmol/L组、1.0μmol/组和2.0μmol/组p16基因表达阳性条带灰度值与β-肌动蛋白比值分别为(0.22±0.10)、(0.59±0.11)、(0.68±0.09),阳性对照灰度比值为(0.77±0.13),差异有非常显著的统计学意义(P<0.01);③与未处理组相比,As2O3作用72小时后甲基转移酶(DNMT)1、DNMT3A、DNMT3B的表达下降并呈浓度依赖性;④与对照组相比,3组不同浓度As2O3均能明显抑制骨髓瘤细胞生长,G0-G1期细胞增加。结论:As2O3可能通过抑制甲基转移酶(DNMT1)、DNMT3A、DNMT3B和(或)直接对p16基因去甲基化,使p16基因表达上调,恢复其活性,从而实现其对细胞周期的调控功能,将细胞阻滞于G0-G1期,抑制骨髓瘤细胞的增长。The study was purposed to investigate the effect of arsenic trioxide（ As2O3 ） - induced p16 gene demethylation by a sensitive and specific PCR-based method （nested-methylation specific PCR, n-MSP） and DNA sequencing for rapid analysis of the promoter demethylation status, and to explore the possible mechanism of the pl6 gene demethylation in human multiple myeloma U266 cells induced by As2O3. The methylation status of the p16 gene in U266 cell line before and after treatment with As2O3 was detected by the nested-methylation specific PCR and DNA sequencing, the mRNA of p16, DNA methyltransferase （DNMT 1 、DNMT3A and 3B） gene were determined by RT-PCR, and the induced growth inhibition of U266 cell was assayed by growth curve, MTT and CFU; the DNA content of U266 cells was analyzed by flow cytometry after being exposed to As2O3. The results showed that （ 1 ） all cytosines in CpG dinucleotides in untreated U266 cell not were changed , while all cytosines in treated U266 cells with As2 O3 had been converted to thymidine. （2） p16 gene was not expressed in U266 cell line after methylation. As compared with the β-actin, the expression of U266 cell p16 gene mRNA was increased to （ 0.22 ± 0. 10 ） 、 （ 0.59 ± 0. 11 ）、 （ 0.68 ± 0.09 ） after exposed to 0.5 μmol/L、 1.0 μmol/L and 2. 0μmol/L As2 O3 for 72 hours respectively . （ 3 ） As2 O3 could significantly down-regulate DNA methyllransferase 1（DNMT 1 ）, DNMT3A and DNMT3B gene at mRNA level in a dose-dependent manner. （4） U266 cells line grew slowly and arrested at Go - G1 phase after treatment with three different concentrations of AS203. It is concluded that As2O3 can activate and up-regulate the expression of p16 gene which inhibits the proliferation of U266 cell through inducing the GO - G1 arrest by demethylation or/and by inhibiting DNMT 1 ,DNMT3A and 3B gene.

关 键 词：巢式MSP p16基因去甲基化 三氧化二砷 多发性骨髓瘤 U266细胞 甲基转移酶 

分 类 号：R733.3[医药卫生—肿瘤] R979.1[医药卫生—临床医学]