Jurkat细胞SFRP基因甲基化及去甲基化诱导凋亡的研究  

Promoter methylation status of SFRP genes and induced apoptosis by demethylation in Jurkat cells

作　　者：徐成波[1] 沈建箴[2] 廖斌[1] 付海英[2] 周华蓉[2] 齐彦[1] 皇甫真萍[1] 陈毅宁[1] 陈佳薇[1] 

机构地区：[1]福建省人民医院、福建中医药大学附属人民医院血液科,福州350004 [2]福建医科大学附属协和医院福建省血液病研究所

出　　处：《中华血液学杂志》2016年第1期51-55,共5页Chinese Journal of Hematology

基　　金：福建省自然科学基金(2015J01327)Fund program:Natural Science Foundation of Fujian Province

摘　　要：目的探讨人T淋巴细胞白血病（T-ALL）细胞株Jurkat细胞分泌性卷曲相关蛋白（SFRP）基因甲基化及去甲基化诱导细胞凋亡对Wnt／β-catenin信号通路的影响。方法以不同浓度（1．0、2．0、4．0gmol／L）5．杂氮．2’-脱氧胞苷（5-Aza．CdR）对Jurkat细胞进行去甲基化处理，采用MTT法观察5-Aza-CdR对Jurkat细胞增殖的抑制作用，流式细胞术检测细胞凋亡率，甲基化特异性PCR（MSP）法检测药物处理前后SFRP基因的甲基化状态，实时荧光定量PCR检测SFRP基因以及RT-PCR检测survivin、C—myc和cyclinDl基因mRNA的表达改变，Westernblot鉴定处理前后13-catenin的蛋白表达。结果1．0、2．0、4．0gmol／L5-Aza-CdR对Jurkat细胞的增殖有明显抑制作用，呈时间-剂量依赖性（P〈0．05）；流式细胞术检测显示5-Aza—CdR作用Jurkat细胞48h后，不同浓度5-Aza—CdR处理组与对照组比较细胞早期凋亡率明显升高（P〈0．05）；SFRP1、SFRP2、SFRP4、SFRP5基因甲基化水平随5-Aza—CdR浓度升高而下降，呈剂量依赖性（P〈0．05），同时mRNA表达水平较对照组明显上调（P〈O．05）；Jurkat细胞总蛋白中13-catenin的蛋白表达随5-Aza-CdR浓度的升高而逐渐下降，呈剂量依赖性（P〈0．05）；凋亡相关基因survivin、C—myc和cyclinD1的mRNA表达随5-Aza-CdR浓度的增高而降低，呈剂量依赖性（P〈0．05）。结论逆转Jurkat细胞SFRP基因的甲基化，可以恢复SFRP基因转录表达，通过阻断13．catenin蛋白抑制Wnt／13-catenin信号通路的激活而诱导细胞凋亡。Objective To study the promoter methylation status of SFRP genes and the effect of 5- aza-2＇- deoxycytidine （5-Aza-CdR） induced apoptosis via Wnt/β- catenin pathway by demethylation in Jurkat cells. Methods Jurkat cells were treated with different concentrations of 5-Aza-CdR. The cell proliferation level of Jurkat cells was detected by MTT assay. Apoptosis was evaluated by flow cytometry. Methylation-spcific PCR （MSP） was used to determine the methylation status of SFRP genes. The expressions of SFRP genes were detected by real time fluorescence quantitative PCR. The mRNA expression levels of survivin, c-myc and cyclin-D1 were analyzed by RT-PCR. Western blot was used to detect the levels of β-catenin protein. Results Compared with control group, the different concentrations of 5-Aza-CdR could significantly inhibit the proliferation of Jurkat cells in a time-dose dependent manner （P 〈 0.05）. After being treated by 5-Aza-CdR for 48 hours, the cell early apoptosis rate in experiment group was significantly higher than that in control group （P〈 0.05）. The promoters of SFRP1, SFRP2, SFRP4, SFRP5 genes were hypermethylation state in the control group, after being treated by 5-Aza-CdR for 72 hours, the brightness of SFRP1, SFRP2, SFRP4, SFRP5 genes＇ methylation strips weakened in a dose-dependent manner. SFRP mRNA expression increased （P 〈 0.05） when 5-Aza-CdR concentration increased, and the level of β-catenin protein was dampened in a dose-dependent manner （P 〈 0.05）. Ascompared to the control group, the mRNA expressions of associated apoptosis genes survivin, c-myc and cyclin-D1, respectively were obviously down-regulated in a dose-dependent manner （P〈0.05）. Conclusion The effect of demetbylation could up-regulate SFRP genes expressions by reversing its hypermethylation and induced apoptosis by down-regulation of β-catenin and associated apoptosis genes.

关 键 词：SFRP基因 DNA甲基化 WNT/Β-CATENIN JURKAT细胞 细胞凋亡 

分 类 号：R733.7[医药卫生—肿瘤]