左旋多巴处理SH-SY5Y细胞可能通过DNA甲基化调节单胺氧化酶B基因的转录

doi:10.3969/j.issn.1006-7795.2015.05.014

首都医科大学学报 ›› 2015, Vol. 36 ›› Issue (5): 734-739.doi: 10.3969/j.issn.1006-7795.2015.05.014

左旋多巴处理SH-SY5Y细胞可能通过DNA甲基化调节单胺氧化酶B基因的转录

杨兆菲^1,2, 王勇^2,3, 杨俭^1,2, 汪璇^2,3, 王晓民^1,2

1. 首都医科大学基础医学院神经生物学系教育部神经变性病重点实验室, 北京 100069;
2. 北京脑重大疾病研究院, 北京 100069;
3. 首都医科大学基础医学院生理学与病理生理学系教育部神经变性病重点实验室, 北京 100069

收稿日期:2015-08-03 出版日期:2015-10-21 发布日期:2015-10-20
基金资助:
国家重点基础研究发展计划(2011CB504100),北京市自然科学基金项目(7082008), 北京市属高等学校创新团队建设与教师职业发展计划项目 (IDHT20140514)。

L-DOPA regulates MAO-B transcription through DNA methylation in SH-SY5Y cells

Yang Zhaofei^1,2, Wang Yong^2,3, Yang Jian^1,2, Wang Xuan^2,3, Wang Xiaomin^1,2

1. Key Laboratory for Neurodegenerative Disorders of the Ministry of Education, Department of Neurobiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China;
2. Beijing Institute for Brain Disorders, Beijing 100069, China;
3. Key Laboratory for Neurodegenerative Disorders of the Ministry of Education, Department of Physiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China

Received:2015-08-03 Online:2015-10-21 Published:2015-10-20
Contact: 王晓民 E-mail:xmwang@ccmu.edu.cn
Supported by:
This study was supported by Major State Basic Research Development Program of China (973 Program)(2011CB504100), Natural Science Foundation of Beijing (7082008), Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges Under Beijing Municipality (IDHT20140514).

摘要/Abstract

摘要： 目的探究左旋多巴(L-3,4-dihydroxyphenylalanine,L-DOPA)对多巴胺代谢关键基因单胺氧化酶B(monoamine oxidase B,MAO-B)转录的影响及其表观遗传学调控机制。方法以人神经母细胞瘤SH-SY5Y细胞作为研究对象,应用基因组甲基化定量试剂盒检测L-DOPA对基因组甲基化的影响;采用半定量RT-PCR方法探究L-DOPA和DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷(5-aza-2'-deoxycytidine,5-aza-dC)对MAO-B转录的影响;应用甲基化特异性PCR(methylation specific PCR,MSP)技术检测L-DOPA对MAO-B基因启动子区CpG岛甲基化程度的影响。结果 1L-DOPA(2 μmol/L和20 μmol/L)和50 μmol/L 5-aza-dC处理SH-SY5Y细胞24 h均会使其基因组甲基化降低。2L-DOPA(2 μmol/L和20 μmol/L)处理SH-SY5Y细胞24 h会使MAO-B mRNA下调,而多巴脱羧酶(DOPA-decarboxylase,DDC)和儿茶酚胺氧位甲基转移酶(catechol-O-methyltransferase,COMT)转录无明显变化。3不同浓度5-aza-dC(0、50、100和150 μmol/L)处理细胞24 h,MAO-B mRNA表达明显上调,DDC和COMT转录无明显变化,提示MAO-B可能受到DNA甲基化调控。4L-DOPA处理可使MAO-B基因启动子区CpG岛甲基化升高,而5-aza-dC可使该区域CpG岛甲基化降低,说明该区域甲基化受L-DOPA影响而升高,并可能是MAO-B基因转录活性下调的原因。结论 L-DOPA可能对MAO-B基因的转录活性有抑制作用,且可能是通过L-DOPA诱导的高CpG甲基化所导致,从表观遗传学(主要是DNA甲基化)对临床上L-DOPA治疗可能产生的多巴胺代谢紊乱及其不良反应做出了新的解释。

关键词: 左旋多巴, 单胺氧化酶B, DNA甲基化, 基因转录

Abstract: Objective To explore the effect and molecular mechanism of L-3,4-dihydroxyphenylalanine (L-DOPA) on transcription of monoamine oxidase B (MAO-B), which was a key gene in dopamine metabolisms. Methods SH-SY5Y cells were used as the object to mimic dopaminergic neurons in Parkinson's disease. Methylated DNA quantitation kit was used to analyze genomic 5-mC content. The gene transcriptional levels were measured by semi-quantitative RT-PCR. Methylation specific PCR (MSP) was applied to detect the promotor CpG methylation status of MAO-B. Results 1 Genomic 5-mC content was decreased in SH-SY5Y cells treated with L-DOPA (2 μmol/L and 20 μmol/L) for 24 h. 2 Monoamine oxidase B (MAO-B) mRNA level was significantly down-regulation in L-dopa administration, but neither DOPA-decarboxylase (DDC) nor catechol-O-methyltransferase (COMT). 3 A DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), induced MAO-B mRNA level up-regulation, but COMT and DDC mRNA levels did not affected. 4 CpG hyper-methylation of MAO-B was induced by L-DOPA (2 μmol/L and 20 μmol/L) for 24 h. And CpG hypo-methylation was induced by 5-aza-dC. Conclusion Transcription of MAO-B was down-regulated by L-DOPA and might be mediated by L-DOPA induced promoter CpG hyper-methylation. This study might provide a new evidence in the view of epigenetics, especially DNA methylation, on dopamine dysregulation and L-DOPA induced side effects in clinic.

Key words: L-3,4-dihydroxyphenylalanine, monoamine oxidase B, DNA methylation, gene transcription

中图分类号:

杨兆菲, 王勇, 杨俭, 汪璇, 王晓民. 左旋多巴处理SH-SY5Y细胞可能通过DNA甲基化调节单胺氧化酶B基因的转录[J]. 首都医科大学学报, 2015, 36(5): 734-739.

Yang Zhaofei, Wang Yong, Yang Jian, Wang Xuan, Wang Xiaomin. L-DOPA regulates MAO-B transcription through DNA methylation in SH-SY5Y cells[J]. Journal of Capital Medical University, 2015, 36(5): 734-739.

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左旋多巴处理SH-SY5Y细胞可能通过DNA甲基化调节单胺氧化酶B基因的转录

L-DOPA regulates MAO-B transcription through DNA methylation in SH-SY5Y cells

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