shRNA沉默LSD1基因对Jurkat细胞增殖的影响及机制研究  

Antiproliferative effect of silencing LSD1 gene on Jurkat cell line and its mechanism

作　　者：韩世炜[1,2] 黄轶群[1] 郑瑞玑[1] 

机构地区：[1]福建医科大学附属漳州市医院血液内科,363000 [2]浙江省奉化市人民医院血液肿瘤科,315500

出　　处：《中华血液学杂志》2016年第1期56-60,共5页Chinese Journal of Hematology

基　　金：福建省卫生系统中青年骨干人才培养项目(2014-ZQN-JC-36)Fund program:Fujian Provincial Health System of Young Backbone Talents Training Project

摘　　要：目的探讨RNA靶向沉默LSDl基因后对急性T淋巴细胞白血病细胞株Jurkat细胞增殖、凋亡的影响及其可能的作用机制。方法将LSDl短发夹RNA（shRNA）真核表达载体经脂质体转染入Jurkat细胞，采用RQ-PCR及Westernblot方法观察LSDlmRNA及蛋白表达；采用MTT法观察细胞增殖情况，采用流式细胞仪检测细胞凋亡情况，Westernblot方法检测凋亡相关蛋白Bc1-2、Bax、procaspase-3的表达及组蛋白H3K4me、H3K4me2、H3K4me3、Act-H3水平。结果LSDlshRNA转染Jurkat细胞后，LSDlmRNA、蛋白表达均下调（P〈0．05）；LSDlshRNA组48h细胞增殖率低于转染阴性质粒（Neg—shRNA）组和对照组（P〈0．05），LSDlshRNA组细胞凋亡率[（41．34±3．58）％]高于Neg-shRNA组[（3．45±1．54）％]和对照组[（1．76±0．52）％]（P〈0．05）；凋亡抑制蛋白Bcl-2、凋亡效应蛋白procaspase-3的表达下调，Bax表达上调，H3K4me、H3K4me2、Act—H3表达上调，而H3K4me3水平未见明显变化。结论沉默LSDl基因可抑制Jurkat细胞增殖，激活凋亡相关蛋白诱导细胞凋亡，机制可能与调节组蛋白H3K4甲基化水平有关．Objective To investigate the effect of silencing LSD1 gene by RNA interference on the proliferation, apoptosis on human lymphocytic leukemia Jurkat cell line and its mechanism. Methods The hairpin-like oligonucleotide sequences targeting LSD1 gene was transfected into Jurkat cells by lipofectamineTM 2000. The LSD1 mRNA and protein were detected by RQ-PCR and Western blot. Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expression of Bcl-2, Bax, procaspase-3, and histone H3K4me, H3K4me2, H3K4me3, Act- H3, H3K9me were detected by Western blot. Results LSD1 mRNA was markedly suppressed by the shRNA targeting LSD1. LSD1 shRNA suppressed the proliferation and induced cells apoptosis of Jurkat cells. The cell apoptotic rate was （41.34±3.58）%, （3.45±1.54）%, （ 1.76±0.52）% in LSD1 shRNA, Neg-shRNA and Blank respectively, the difference among them was statistically significant （P〈0.05）. LSD1 shRNA downregulated the expressions of Bcl- 2 and procaspase- 3, and upregulated the expression of Bax. The methylation of H3K4mel, me2 and acetylation of Act-H3 improved without change of the methylation of H3K4me3. Conclusions Deplete of LSD1 gene maybe through modifying the methylation of histone H3K4 to promote the cell apoptosis and inhibit cell growth in Jurkat cell line.

关 键 词：赖氨酸特异性组蛋白去甲基化酶1 RNA干扰 JURKAT细胞 组蛋白类 

分 类 号：R733.7[医药卫生—肿瘤]