药学学报, 2017, 52(1): 91-98
引用本文:
何航, 王沛, 李世刚, 陈钰龙, 阚全程, 张莉蓉. 优降宁对CYP3A4/3A7基因启动子和增强子区组蛋白甲基化修饰及其转录的影响[J]. 药学学报, 2017, 52(1): 91-98.
HE Hang, WANG Pei, LI Shi-gang, CHEN Yu-long, KAN Quan-cheng, ZHANG Li-rong. Effects of pargyline on histone methylation in promoter and enhancer regions and transcription of CYP3A4/3A7[J]. Acta Pharmaceutica Sinica, 2017, 52(1): 91-98.

优降宁对CYP3A4/3A7基因启动子和增强子区组蛋白甲基化修饰及其转录的影响
何航1,3, 王沛1, 李世刚1, 陈钰龙2, 阚全程2, 张莉蓉1
1. 郑州大学基础医学院, 河南 郑州 450001;
2. 郑州大学第一附属医院, 河南 郑州 450052;
3. 河南中医药大学, 河南 郑州 450046
摘要:
观察优降宁(pargyline)对细胞色素P450 3A(cytochrome P450 3A,CYP3A)亚型CYP3A4/3A7启动子及增强子区组蛋白甲基化修饰及其基因转录的影响。原代分离培养人胎肝细胞,分为空白对照组、溶媒组、优降宁低、中、高(0.6、1.2、2.4 mmol·L-1)浓度组,HepG2细胞用0.03、0.3、3 mmol·L-1优降宁处理48 h,观察组蛋白甲基化修饰对CYP3A4/3A7基因表达的影响;3 mmol·L-1优降宁处理组与对照组的HepG2细胞进行染色质免疫共沉淀,选取特异CYP3A4/3A7启动子、增强子区位点设计引物进行实时定量PCR,H3K4me2富集以input百分比表示观察富集的改变。结果表明,优降宁能促进人原代胎肝细胞、HepG2细胞增殖生长,与溶媒对照组相比,优降宁(1.2、2.4 mmol·L-1)显著升高人原代胎肝细胞CYP3A7表达水平,且优降宁(3 mmol·L-1)显著升高HepG2细胞CYP3A4/3A7表达水平(P<0.001);CYP3A4近端启动子区(-362~+53)和增强子区(-7 836~-6 093)的肝细胞核因子4A(hepatocyte nuclear factors 4A,HNF4A)结合位点及CYP3A7启动子区(-163~+103)和增强子区(-4 054~-3 421,-6 265~-6 247)的糖皮质激素受体(glucocorticoid receptor,GR)结合位点与对照组相比,处理组均表现为H3K4me2高度修饰(P<0.01,P<0.001)。提示优降宁诱导CYP3A4/3A7启动子及增强子区HNF4A、GR结合位点H3K4me2高度富集激活CYP3A4/3A7基因转录。
关键词:    个体发育      细胞色素P450 3A4      表观遗传      组蛋白甲基化      核受体      优降宁     
Effects of pargyline on histone methylation in promoter and enhancer regions and transcription of CYP3A4/3A7
HE Hang1,3, WANG Pei1, LI Shi-gang1, CHEN Yu-long2, KAN Quan-cheng2, ZHANG Li-rong1
1. School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China;
2. The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China;
3. Henan University of Chinese Medicine, Zhengzhou 450046, China
Abstract:
This study was designed to investigate effects of pargyline on histone methylation in the promoter and enhancer regions and transcription of cytochrome P450 3A4/3A7 (CYP3A4/3A7) gene. Human primary fetal liver cells were isolated, cultured and randomly divided into several groups including control, solvent, pargyline low, middle, high dose (treated with 0.6, 1.2, 2.4 mmol·L-1). HepG2 cells were cultured and treated with 0.03, 0.3, 3 mmol·L-1 pargyline. After 48 hours, total RNAs were prepared from the cells to determine the expression of CYP3A mRNA in primary fetal cells and HepG2 cells with real-time quantative PCR (qPCR). HepG2 cells were cultured and then treated with 3 mmol·L-1 pargyline for 48 hours. The chromatin immunoprecipitation (ChIP) assay was performed with dimethylation of histone H3 at lysine 4 (H3K4me2), and IgG antibodies respectively. The precipitated DNA was resuspended and used for qPCR. Primers were used to detect different regions of CYP3A4/3A7 promoter and enhancer. Occupancy of H3K4me2 was shown as percent of input DNA relative to control cells. The results suggested that pargyline has an effect on primary fetal liver cells and HepG2 cells proliferation. The level of CYP3A7 was markedly enhanced in human primary fetal liver cells by treatment with 1.2, 2.4 mmol·L-1 of pargyline (P<0.05, P <0.01) and the levels of CYP3A4/3A7 were remarkably enhanced by treatment with 3 mmol·L-1 of pargyline in HepG2 cells (P<0.001) compared with solvent control. Occupancy of H3K4me2 on human CYP3A4 promoter (-362 to +53) and enhancer segment (-7 836 to -6 093) harbored the overlapping hepatocyte nuclear factors 4A (HNF4A) binding site compared with a negative control. Occupancy of H3K4me2 on human CYP3A7 promoter (-163 to +103) and enhancer segment (-4 054 to -3 421, -6 265 to -6 247) overlapped with glucocorticoid receptor (GR) binding site. In conclusion, the enriched H3K4me2 in the promoter and enhancer regions was induced by pargyline with HNF4A or GR binding site in CYP3A4/3A7 gene to activate the corresponding genes.
Key words:    ontogeny    cytochrome P450 3A4    epigenetics    histone methylation    nuclear receptor    pargyline   
收稿日期: 2016-08-03
DOI: 10.16438/j.0513-4870.2016-0767
基金项目: 国家自然科学基金资助项目(81173127,81273581).
通讯作者: 张莉蓉,Tel:86-371-67781855,Fax:86-371-66913510,E-mail:zhanglirongzzu@126.com
Email: zhanglirongzzu@126.com
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