内侧前额叶皮质DNA甲基化调控大鼠酒精相关行为

doi:10.16288/j.yczz.19-261

遗传 ›› 2020, Vol. 42 ›› Issue (1): 112-125.doi: 10.16288/j.yczz.19-261

内侧前额叶皮质DNA甲基化调控大鼠酒精相关行为

崔亨贞¹, 孙蜜烛², 王润芝², 李辰雨³, 黄予暄³, 黄秋菊³, 乔晓孟²()

^1. 郑州大学医学院基础医学专业,郑州 450001
^2. 郑州大学医学院法医学系,郑州 450001
^3. 郑州大学医学院医学检验系,郑州 450001

收稿日期:2019-09-02 修回日期:2019-11-21 出版日期:2020-01-20 发布日期:2019-12-25
通讯作者: 乔晓孟 E-mail:xiaomeng416520@126.com
作者简介:崔亨贞,本科生,专业方向：基础医学。E-mail: chzzzu@163.com
基金资助:
国家自然科学基金项目编号(81373247);郑州大学大学生创新项目资助编号(2019cxcy054)

DNA methylation in the medial prefrontal cortex regulates alcohol-related behavior in rats

Hengzhen Cui¹, Mizhu Sun², Runzhi Wang², Chenyu Li³, Yuxuan Huang³, Qiuju Huang³, Xiaomeng Qiao²()

^1. Basic Medicine, School of Medicine, Zhengzhou University, Zhengzhou 450001, China
^2. Department of Forensic Medicine, School of Medicine, Zhengzhou University, Zhengzhou 450001, China
^3. Department of Medical Laboratory,School of Medicine, Zhengzhou University, Zhengzhou 450001, China

Received:2019-09-02 Revised:2019-11-21 Online:2020-01-20 Published:2019-12-25
Contact: Qiao Xiaomeng E-mail:xiaomeng416520@126.com
Supported by:
Supported by the National Natural Science Foundation of China No(81373247);the Innovative Project for College Students of Zhengzhou University No(2019cxcy054)

摘要/Abstract

摘要：

酒精滥用不仅导致组织器官损伤,还易诱发神经精神疾病。研究表明,DNA甲基化在酒精诱导基因表达和行为改变中发挥重要作用,但具体的神经生物学机制尚未被阐明。为了探索DNA甲基化在酒精滥用中的作用机制,本研究选取健康成年雄性 SD大鼠(Rattus norvegicus) 32只,随机分为饮水对照组(n=16)和慢性酒精暴露组(n=16),运用双瓶选择实验(two bottle choice test, TBCT)评估大鼠酒精偏爱率(alcohol preference),通过旷场行为(open field test, OFT)评估活动状态并检测血酒精浓度。分离两组大鼠内侧前额叶皮质(medial prefrontal cortex, mPFC),提取总DNA,利用简化代表性重亚硫酸盐测序技术(reduced representation bisulfite sequencing, RRBS)构建mPFC甲基化谱,对差异基因进行功能富集和通路分析,筛选与酒精滥用密切相关的甲基化差异基因,运用qRT-PCR技术检测差异基因的表达,验证DNA甲基化对基因的表达调控;利用qRT-PCR和Western blot检测甲基转移酶(DNA methyltransferases, DNMTs)和甲基化CpG 位点结合蛋白2 (methyl CpG binding protein 2, MeCP2)的表达;同时,还检测了短期酒精暴露(7 d)对大鼠mPFC内DNMTs和MeCP2的影响(n=8/组)。结果表明,慢性酒精暴露大鼠mPFC内基因启动子区甲基化水平显著升高。与酒精滥用密切相关的差异基因中,慢性酒精暴露组Ntf3和Ppm1G启动子区甲基化水平升高,mRNA表达降低;Hap1和DUSP1启动子区甲基化水平降低,mRNA表达升高。慢性酒精暴露使DNMT3B和MeCP2 mRNA和蛋白表达升高,而短期内酒精暴露不影响它们的表达。本研究初步证实DNA甲基化与酒精滥用的发展相关,可能受DNMT3B和MeCP2分子的调控,并发现了与酒精滥用相关的靶基因Ntf3、Ppm1G、Hap1和DUSP1,为研究酒精滥用的神经生物学机制提供了新见解,同时为酒精滥用治疗提供了可能的药理学靶点。

关键词: 酒精滥用, DNA甲基化, DNA甲基转移酶, 内侧前额叶皮质

Abstract:

Alcohol abuse causes tissue and organ damage, and may participate neuropsychiatric diseases. Studies have shown that DNA methylation plays an important role in gene expression and behavioral changes induced by alcohol, however the causative neurobiological mechanisms have not been clarified. In this study, 32 healthy adult male SD rats were randomly divided into a drinking water control group (n=16) and a chronic alcohol exposure group (n=16). The alcohol preference and locomotor activity of rats were evaluated by two-bottle choice test (TBCT) and open-field test (OFT). DNA methylation in the medial prefrontal cortex (mPFC) tissue was detected by the reduced representative bisulfite sequencing (RRBS) technology. The methylation differential genes closely related to alcohol abuse were screened. qRT-PCR was used to verify the mRNA expression patterns of differential genes. qRT-PCR and Western blot were used to detect the expression of DNA methyltransferases (DNMTs) and methyl CpG binding protein 2 (MeCP2). Furthermore, the effect of short-term alcohol exposure (7 days) on DNMTs and MeCP2 in the mPFC of rats was tested (n=8/group). The results indicated that the methylation level of promoter region in the mPFC of rats exposed to chronic alcohol was significantly increased. In addition, the increased methylation levels in the promoter of Ntf3 and Ppm1G were accompanied by down-regulated mRNA levels in the chronic alcohol exposure group. The decreased methylation levels in the promoter of Hap1 and DUSP1 were accompanied by up-regulated mRNA levels. Furthermore, chronic alcohol exposure increased the mRNA and protein levels of DNMT3B and MeCP2. However, short term alcohol exposure did not affect their expression. This present study provides evidence that DNA methylation is associated with the development of alcohol abuse, which may be regulated by DNMT3B and MeCP2. The target genes Ntf3, Ppm1G, Hap1, and DUSP1 related to alcohol abuse were discovered as well, providing new insights into the neurobiological mechanism of alcohol abuse and the potential pharmacological targets for the treatment of alcohol abuse.

Key words: alcohol abuse, DNA methylation, DNA methyltransferases, medial prefrontal cortex

崔亨贞, 孙蜜烛, 王润芝, 李辰雨, 黄予暄, 黄秋菊, 乔晓孟. 内侧前额叶皮质DNA甲基化调控大鼠酒精相关行为[J]. 遗传, 2020, 42(1): 112-125.

Hengzhen Cui, Mizhu Sun, Runzhi Wang, Chenyu Li, Yuxuan Huang, Qiuju Huang, Xiaomeng Qiao. DNA methylation in the medial prefrontal cortex regulates alcohol-related behavior in rats[J]. Hereditas(Beijing), 2020, 42(1): 112-125.

图/表 7

表1

图1

图2

图3

图4

图5

图6

参考文献 33

[1]	Peacock A, Leung J, Larney S, Colledge S, Hickman M, Rehm J, Giovino GA, West R, Hall W, Griffiths P, Ali R, Gowing L, Marsden J, Ferrari AJ, Grebely J, Farrell M, Degenhardt L . Global statistics on alcohol, tobacco and illicit drug use: 2017 status report. Addiction, 2018,113(10):1905-1926. doi: 10.1111/add.14234 pmid: 29749059
[2]	WHO. Global status report on alcohol and health. World Health Organization, 2014. doi: 10.1016/j.abrep.2019.100216 pmid: 31692685
[3]	Lu SJ, Du SM, Hu XQ, Zou SR, Liu WJ, Ba L, Ma GS . Drinking patterns and the association between socio- demographic factors and adolescents' alcohol use in three metropolises in China. Int J Environ Res Public Health, 2015,12(2):2037-2053. doi: 10.3390/ijerph120202037 pmid: 25685952
[4]	Antonelli M, Ferrulli A, Sestito L, Vassallo GA, Tarli C, Mosoni C, Rando MM, Mirijello A, Gasbarrini A, Addolorato G . Alcohol addiction-the safety of available approved treatment options. Expert Opin Drug Saf, 2018,17(2):169-177. doi: 10.1080/14740338.2018.1404025 pmid: 29120249
[5]	Yang X, Zhang H, Lai J . Alcohol dependence mediated by monoamine neurotransmitters in the central nervous system. Hereditas (Beijing), 2014,36(1):11-20. doi: 10.3724/SP.J.1005.2014.00011
	杨晓华, 张华峰, 赖江华 . 中枢单胺类神经递质在酒精依赖中的分子作用机制. 遗传, 2014,36(1):11-20. doi: 10.3724/SP.J.1005.2014.00011
[6]	He GF, Zhong SR, Jing Q . Genetic polymorphism for genes of alcohol dependence. Hereditas (Beijing), 2008,30(4):413-418. doi: 10.3724/SP.J.1005.2008.00413
	贺艮峰, 钟树荣, 景强 . 酒精依赖相关基因的遗传多态性. 遗传, 2008,30(4):413-418. doi: 10.3724/SP.J.1005.2008.00413
[7]	Nestler EJ . Epigenetic mechanisms of drug addiction. Neuropharmacology, 2014,76:259-268. doi: 10.1016/j.neuropharm.2013.04.004
[8]	Zhu ZZ, Hou LF, Bollati V, Tarantini L, Marinelli B, Cantone L, Yang AS, Vokonas P, Lissowska J, Fustinoni S, Pesatori AC, Bonzini M, Apostoli P, Costa G, Bertazzi PA, Chow WH, Schwartz J, Baccarelli A . Predictors of global methylation levels in blood DNA of healthy subjects: a combined analysis. Int J Epidemiol, 2012,41(1):126-139. doi: 10.1093/ije/dyq154
[9]	Ruggeri B, Nymberg C, Vuoksimaa E, Lourdusamy A, Wong CP, Carvalho FM, Jia T, Cattrell A, Macare C, Banaschewski T, Barker GJ, Bokde AL, Bromberg U, Büchel C, Conrod PJ, Fauth-Bühler M, Flor H, Frouin V, Gallinat J, Garavan H, Gowland P, Heinz A, Ittermann B, Martinot JL, Nees F, Pausova Z, Paus T, Rietschel M, Robbins T, Smolka MN, Spanagel R, Bakalkin G, Mill J, Sommer WH, Rose RJ, Yan J, Aliev F, Dick D, Kaprio J, Desrivières S, Schumann G . Association of protein phosphatase PPM1G with alcohol use disorder and brain activity during behavioral control in a genome-wide methylation analysis. Am J Psychiatry, 2015,172(6):543-552. doi: 10.1176/appi.ajp.2014.14030382 pmid: 25982659
[10]	Barker JM, Zhang YQ, Wang F, Taylor JR, Zhang HP . Ethanol-induced Htr3a promoter methylation changes in mouse blood and brain. Alcohol Clin Exp Res, 2013,37(s1):E101-E107. doi: 10.1111/j.1530-0277.2012.01906.x pmid: 22834954
[11]	Barbier E, Tapocik JD, Juergens N, Pitcairn C, Borich A, Schank JR, Sun H, Schuebel K, Zhou Z, Yuan Q, Vendruscolo LF, Goldman D, Heilig M . DNA methylation in the medial prefrontal cortex regulates alcohol-induced behavior and plasticity. . Neurosci, 2015,35(15):6153-6164. doi: 10.1523/JNEUROSCI.4571-14.2015 pmid: 25878287
[12]	Qiao XM, Yin FY, Ji YY, Li YX, Yan P, Lai JH . 5-Aza-2'-deoxycytidine in the medial prefrontal cortex regulates alcohol-related behavior and Ntf3-TrkC expression in rats. PLoS One, 2017,12(6):e0179469. doi: 10.1371/journal.pone.0179469 pmid: 28614398
[13]	Boerngen-Lacerda R, Souza-Formigoni ML . Does the increase in locomotion induced by ethanol indicate its stimulant or anxiolytic properties? Pharmacol Biochem Behav, 2000,67(2):225-232. doi: 10.1016/s0091-3057(00)00360-9 pmid: 11124385
[14]	Boschen KE, Hamilton GF, Delorme JE, Klintsova AY . Activity and social behavior in a complex environment in rats neonatally exposed to alcohol. Alcohol 2014; 48:533-541. doi: 10.1016/j.alcohol.2014.07.005
[15]	Buckner JD, Terlecki MA . Social anxiety and alcohol- related impairment: The mediational impact of solitary drinking. Addict Behav, 2016,58:7-11. doi: 10.1016/j.addbeh.2016.02.006 pmid: 26894561
[16]	Colombo G, Lobina C, Lorrai I, Acciaro C, Maccioni P, Gessa GL . Binge drinking and anxiety at the end of the nocturnal period in alcohol-preferring sP rats. Alcohol, 2017,63:27-32. doi: 10.1016/j.alcohol.2017.04.002 pmid: 28847379
[17]	Thorberg FA, Young RM, Hasking P, Lyvers M, Connor JP, London ED, Huang YL, Feeney GFX . Alexithymia and alcohol dependence: The roles of negative mood and alcohol craving. Subst Use Misuse, 2019,54(14):1-7. doi: 10.1080/10826084.2019.1650773 pmid: 31429362
[18]	Charlet K, Heinz A . Harm reduction-a systematic review on effects of alcohol reduction on physical and mental symptoms. Addict Biol, 2017,22(5):1119-1159. doi: 10.1111/adb.12414 pmid: 27353220
[19]	Bönsch D, Lenz B, Fiszer R, Frieling H, Kornhuber J, Bleich S . Lowered DNA methyltransferase (DNMT-3b) mRNA expression is associated with genomic DNA hypermethylation in patients with chronic alcoholism. . Neural Transm (Vienna), 2006,113(9):1299-1304. doi: 10.1007/s00702-005-0413-2 pmid: 16463117
[20]	Bönsch D, Lenz B, Reulbach U, Kornhuber J, Bleich S . Homocysteine associated genomic DNA hypermethylation in patients with chronic alcoholism. . Neural Transm (Vienna), 2004,111(12):1611-1616. doi: 10.1007/s00702-004-0232-x pmid: 15565495
[21]	Weng JT, Wu LS, Lee CS, Hsu PW, Cheng AT . Integrative epigenetic profiling analysis identifies DNA methylation changes associated with chronic alcohol consumption. Comput Biol Med, 2015,64:299-306. doi: 10.1016/j.compbiomed.2014.12.003 pmid: 25555412
[22]	Usui N, Watanabe K, Ono K, Tomita K, Tamamaki N, Ikenaka K, Takebayashi H . Role of motoneuron-derived neurotrophin 3 in survival and axonal projection of sensory neurons during neural circuit formation. Development, 2012,139(6):1125-1132. doi: 10.1242/dev.069997 pmid: 22318233
[23]	Han KA, Woo D, Kim S, Choii G, Jeon S, Won SY, Kim HM, Heo WD, Um JW, Ko J . Neurotrophin-3 regulates synapse development by modulating trkc-ptpσ synaptic adhesion and intracellular signaling pathways. . Neurosci, 2016,36(17):4816-4831. doi: 10.1523/JNEUROSCI.4024-15.2016 pmid: 27122038
[24]	Tang TS, Tu HP, Chan EYW, Maximov A, Wang ZN, Wellington CL, Hayden MR, Bezprozvanny I . Huntingtin and huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1. Neuron, 2003,39(2):227-239. doi: 10.1016/s0896-6273(03)00366-0 pmid: 12873381
[25]	Czeredys M, Vigont VA, Boeva VA, Mikoshiba K, Kaznacheyeva EV, Kuznicki J . Huntingtin-associated protein 1a regulates store-operated calcium entry in medium spiny neurons from transgenic YAC128 mice, a model of huntington's disease. Front Cell Neurosci, 2018,12:381. doi: 10.3389/fncel.2018.00381 pmid: 30455632
[26]	Kim SH, Yu HS, Park HG, Park S, Seo MS, Jeon WJ, Ahn YM, Ha K, Shin SY, Kim YS . Role of MKP-1 (DUSP1) in clozapine-induced effects on the ERK1/2 signaling pathway in the rat frontal cortex. Psychopharmacology (Berl), 2013,230:425-437. doi: 10.1007/s00213-013-3165-y pmid: 23771439
[27]	Turek-Plewa J, Jagodziński PP . The role of mammalian DNA methyltransferases in the regulation of gene expression. Cell Mol Biol Lett, 2005,10(4):631-647. pmid: 16341272
[28]	Dajun D . DNA methylation and demethylation: current status and future perspective. Hereditas (Beijing), 2014,36(5):403-410. pmid: 24846989
	邓大君 . DNA甲基化和去甲基化的研究现状及思考. 遗传, 2014,36(5):403-410. pmid: 24846989
[29]	Heyward FD, Sweatt JD . DNA methylation in memory formation: emerging insights. Neuroscientist, 2015,21(5):475-489. doi: 10.1177/1073858415579635 pmid: 25832671
[30]	Feng J, Zhou Y, Campbell SL, Le T, Li E, Sweatt JD, Silva AJ, Fan GP . Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat Neurosci, 2010,13(4):423-430. doi: 10.1038/nn.2514 pmid: 20228804
[31]	LaPlant Q, Vialou V, Covington HE, Dumitriu D, Feng J, Warren BL, Maze I, Dietz DM, Watts EL, Iñiguez SD, Koo JW, Mouzon E, Renthal W, Hollis F, Wang H, Noonan MA, Ren Y, Eisch AJ, Bolaños CA, Kabbaj M, Xiao G, Neve RL, Hurd YL, Oosting RS, Fan G, Morrison JH, Nestler EJ . Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens. Nat Neurosci, 2010,13(9):1137-1143. doi: 10.1038/nn.2619 pmid: 20729844
[32]	Repunte-Canonigo V, Chen J, Lefebvre C, Kawamura T, Kreifeldt M, Basson O, Roberts AJ, Sanna PP . MeCP2 regulates ethanol sensitivity and intake. Addict Biol, 2014,19(5):791-799. doi: 10.1111/adb.12047
[33]	Zhang HP, Gelernter J . Review: DNA methylation and alcohol use disorders: Progress and challenges. Am J Addict, 2017,26(5):502-515. doi: 10.1111/ajad.12465 pmid: 27759945

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

基因名称	引物序列(5′→3′)
Ntf3	F: GGCAACAGAGACGCACAAT
Ntf3	R: TCCTCCGTGGTGATGTTCTA
Pdp1	F: CTCAATTACCTGGTGCTTCG
Pdp1	R: GCATTGTGGTCATTAGAGAGC
Ppm1G	F: TCATTGCTTGTGACGGCATC
Ppm1G	R: ATGACAATAACCGAAGCTCCC
FLT1	F: TAACGCAATCTTCTTAACGTC
FLT1	R: TTAATCCTCACAAACGTCCC
N1g2	F: CAGAAGAGACAGAGCAGGCATC
N1g2	R: GCAGAGACCACCACATTCATC
Hap1	F: CAAGGATGAATGTGGTGGTCTC
Hap1	R: CTGCTGCCGCTTAGAATGG
NFκB-p100	F: TCGACCTCCACCGGATCTTT
NFκB-p100	R: CTGTGTCTTTTGGCTTCGGC
RTKN2	F: CCATAGGATGCTAATCTCCGTTT
RTKN2	R: AAGATTACTGCGGTCAACCTT
DUSP1	F: CATTCCCGATGACATACACGTT
DUSP1	R: CATTCCCGATGACATACACGTT
DUSP8	F: TCACTGGTCTGGCTGGCATTTC
DUSP8	R: AGTTGAAGTTGGGCGAGATGG
DNMT1	F: GTTCCTTGTAGGCGAGTGTG
DNMT1	R: TTGCGTAGTCCTGGCTGTAC
DNMT3A	F: GCA AAGTGAGGACCATTACC
DNMT3A	R: GCCAAACACCCTTTCCAT
DNMT3B	F: GTGCGTCGTTCAGGCAGTAG
DNMT3B	R: GTTCTCGGCTCTCCTCATCT
MeCP2	F: GCCTTCAGCCCACCATTCTG
MeCP2	R: CGGTCACGAATGATGGAACG
GAPDH	F: ATGGGGAAGGTGAAGGTCG
GAPDH	R: GGGGTCATTGATGGCAACAA

内侧前额叶皮质DNA甲基化调控大鼠酒精相关行为

DNA methylation in the medial prefrontal cortex regulates alcohol-related behavior in rats

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 33

相关文章 15

编辑推荐

Metrics

[1]	许梦萱, 周明. 植物RNA聚合酶IV调控DNA甲基化和发育的研究进展[J]. 遗传, 2022, 44(7): 567-580.
[2]	王雅楠, 徐涛, 王万鹏, 张庆祝, 解莉楠. 表观遗传修饰在作物重要性状形成中的作用[J]. 遗传, 2021, 43(9): 858-879.
[3]	张向前, 李楠, 解新明. 表观遗传学综合性实验设计与探讨[J]. 遗传, 2021, 43(12): 1179-1187.
[4]	王芯悦, 李亮, 段秋慧, 李大力, 陈金联. Uhrf1对肠上皮发育的影响[J]. 遗传, 2021, 43(1): 84-93.
[5]	王昕源, 张雨, 杨楠, 程禾, 孙玉洁. DNMT3a通过提升基因内部甲基化介导紫杉醇诱导的LINE-1异常表达[J]. 遗传, 2020, 42(1): 100-111.
[6]	黄鑫,陈永强,徐国良,彭淑红. 脂肪组织DNA甲基化与糖尿病和肥胖的发生发展[J]. 遗传, 2019, 41(2): 98-110.
[7]	杨旭琼, 吴珍芳, 李紫聪. 哺乳动物体细胞核移植表观遗传重编程研究进展[J]. 遗传, 2019, 41(12): 1099-1109.
[8]	潘云枫, 王演怡, 陈静雯, 范怡梅. 线粒体代谢介导的表观遗传改变与衰老研究[J]. 遗传, 2019, 41(10): 893-904.
[9]	鞠君毅,赵权. γ-珠蛋白基因表达调控机制与临床应用[J]. 遗传, 2018, 40(6): 429-444.
[10]	刘辰东, 杨露, 蒲红州, 杨琼, 黄文耀, 赵雪, 朱砺, 张顺华. 运动对骨骼肌基因表达的表观遗传调控作用[J]. 遗传, 2017, 39(10): 888-896.
[11]	张轲, 冯光德, 张宝云, 向伟, 陈龙, 杨芳, 储明星, 王凭青. 表观遗传标记在猪分子育种中的研究与应用前景[J]. 遗传, 2016, 38(7): 634-643.
[12]	朱屹然,张美玲,翟志超,赵云蛟,马馨. 生殖细胞及早期胚胎基因组印记的表观调控[J]. 遗传, 2016, 38(2): 103-108.
[13]	刘姝丽，张胜利，俞英. 同卵双胞胎在复杂性状DNA甲基化调控机制研究中的应用[J]. 遗传, 2016, 38(12): 1043-1055.
[14]	刘洋洋, 崔恒宓. DNA甲基化分析中重亚硫酸盐处理DNA转化效率的评估方法[J]. 遗传, 2015, 37(9): 939-944.
[15]	谢龙祥, 于召箫, 郭思瑶, 李萍, AbualgasimElgailiAbdalla, 谢建平. 表观遗传和蛋白质翻译后修饰在细菌耐药中的作用[J]. 遗传, 2015, 37(8): 793-800.