Department of Neuroscience and Experimental Therapeutics , College of Medicine, Texas A&M Health Science Centre, Bryan, TX 77807-3260, USA.
Alcohol Clin Exp Res. 2012 Oct;36(10):1669-77. doi: 10.1111/j.1530-0277.2012.01793.x. Epub 2012 Mar 28.
Ethanol (EtOH) and nicotine are often co-abused. However, their combined effects on fetal neural development, particularly on fetal neural stem cells (NSCs), which generate most neurons of the adult brain during the second trimester of pregnancy, are poorly understood. We previously showed that EtOH influenced NSC maturation in part, by suppressing the expression of specific microRNAs (miRNAs). Here, we tested in fetal NSCs the extent to which EtOH and nicotine coregulated known EtOH-sensitive (miR-9, miR-21, miR-153, and miR-335), a nicotine-sensitive miRNA (miR-140-3p), and mRNAs for nicotinic acetylcholine receptor (nAChR) subunits. Additionally, we tested the extent to which these effects were nAChR dependent.
Gestational day 12.5 mouse fetal murine cerebral cortical-derived neurosphere cultures were exposed to EtOH, nicotine, and mecamylamine, a noncompetitive nAChR antagonist, individually or in combination, for short (24 hour) and long (5 day) periods, to mimic exposure during the in vivo period of neurogenesis. Levels of miRNAs, miRNA-regulated transcripts, and nAChR subunit mRNAs were assessed by quantitative reverse transcription polymerase chain reaction.
EtOH suppressed the expression of known EtOH-sensitive miRNAs and miR-140-3p, while nicotine at concentrations attained by cigarette smokers induced a dose-related increase in these miRNAs. Nicotine's effect was blocked by EtOH and by mecamylamine. Finally, EtOH decreased the expression of nAChR subunit mRNAs and, like mecamylamine, prevented the nicotine-associated increase in α4 and β2 nAChR transcripts.
EtOH and nicotine exert mutually antagonistic, nAChR-mediated effects on teratogen-sensitive miRNAs in fetal NSCs. These data suggest that concurrent exposure to EtOH and nicotine disrupts miRNA regulatory networks that are important for NSC maturation.
乙醇（EtOH）和尼古丁经常同时滥用。然而，它们对胎儿神经发育的联合影响，特别是对妊娠中期第二阶段产生成人大脑大部分神经元的胎儿神经干细胞（NSCs）的影响，了解甚少。我们之前的研究表明，乙醇通过抑制特定 microRNA（miRNA）的表达来影响 NSC 的成熟。在这里，我们在胎儿 NSCs 中测试了乙醇和尼古丁共同调节已知的乙醇敏感（miR-9、miR-21、miR-153 和 miR-335）、尼古丁敏感 miRNA（miR-140-3p）和烟碱型乙酰胆碱受体（nAChR）亚基的程度。此外，我们还测试了这些作用在多大程度上依赖于 nAChR。
妊娠第 12.5 天的小鼠胎儿鼠大脑皮质来源的神经球培养物分别或联合暴露于乙醇、尼古丁和非竞争性 nAChR 拮抗剂美加仑胺中，时间短暂（24 小时）和较长（5 天），以模拟体内神经发生期间的暴露。通过定量逆转录聚合酶链反应评估 miRNA、miRNA 调节的转录物和 nAChR 亚基 mRNA 的水平。
乙醇抑制了已知的乙醇敏感 miRNA 和 miR-140-3p 的表达，而吸烟者体内尼古丁浓度诱导这些 miRNA 的剂量依赖性增加。尼古丁的作用被乙醇和美加仑胺阻断。最后，乙醇降低了 nAChR 亚基 mRNA 的表达，并且与美加仑胺一样，阻止了尼古丁相关的α4 和β2 nAChR 转录物的增加。
乙醇和尼古丁在胎儿 NSCs 中对致畸敏感 miRNA 产生相互拮抗的 nAChR 介导的作用。这些数据表明，同时暴露于乙醇和尼古丁会破坏对 NSC 成熟很重要的 miRNA 调节网络。
