Ethanol alters microNA expression in zebrafish embryos. Danio rerio

Prenatal exposure to ethanol leads to a myriad of developmental disorders known as fetal alcohol spectrum disorder, often characterized by growth and mental retardation, central nervous system damage and specific craniofacial dysmorphic features. Although the exact mechanisms of ethanol toxicity are not well understood it is known that ethanol exposure during development affects the expression of several genes involved in cell cycle control, apoptosis and transcription. MicroRNAs (miRNAs) are implicated in some of these processes however it is unclear if they are involved in ethanol-induced toxicity. Here we tested whether ethanol deregulates miRNA expression in zebrafish embryos and if a miRNA deregulation signature could be inferred. For this, zebrafish embryos were exposed to two different ethanol concentrations (1% and 1.5%) from 4 hours post-fertilization (hpf) to 24hpf. MicroRNA expression profiles revealed that ethanol exposure induces deregulation of miRNA expression significantly. Seven miRNAs are commonly up-regulated after both ethanol treatments, namely miR-153a, miR-725, miR-30d, let-7k, miR-100, miR-738 and miR-732, whereas downregulation of miR-23a, miR-203, let-7c, miR-128 and miR-193b is detected after 1% ethanol exposure only. Target prediction of deregulated miRNAs shows that putative targets are involved in cell cycle control, apoptosis and transcription, which are the main processes affected by ethanol toxicity. The overall study shows that the effects of ethanol on miRNA deregulation are dose-dependent and that miRNAs are relevant in the context of alcohol toxicity. Moreover, a miRNA toxicity signature for embryonic ethanol exposure was obtained. Overall design: Zebrafish embryos were obtained from spawning adults in groups of about 10 males and 10 females. Zebrafish embryos were collected and Petri dishes with approximately 250 eggs each were incubated at 28ºC to allow normal zebrafish development until 4hpf, when blastula is reached. At this stage, embryos were examined under a dissecting microscope and those that had developed normally were selected for EtOH exposure (approximately 200 eggs). Briefly, 200 embryos were randomly distributed into plastic Petri dishes containing 20 mL of EtOH test solutions (1% EtOH, 1.5% EtOH). All solutions were made by dilution of absolute EtOH in system water. Exposure was from 4hpf to 24hpf. At this stage, solutions were changed by system water and embryos were allowed to grow until 24hpf. The control group was allowed to grow in plain system water. Zebrafish embryos were collected at 24hpf for microarray analysis. Two biological replicates were performed for each assay.

产前乙醇暴露会引发一系列被称为胎儿酒精谱系障碍（fetal alcohol spectrum disorder）的发育异常病症，这类病症通常表现为生长发育与智力迟缓、中枢神经系统损伤以及特征性颅面畸形。虽然乙醇毒性的确切致病机制尚未完全阐明，但已有研究证实，发育阶段的乙醇暴露会影响诸多参与细胞周期调控、细胞凋亡及转录过程的基因的表达。微小RNA（microRNAs，miRNAs）参与了上述部分生理过程，但目前尚不明确其是否参与乙醇诱导的毒性反应。本研究旨在探究乙醇是否会诱导斑马鱼胚胎的miRNA表达失调，以及能否推导得到miRNA失调特征谱。为此，本研究于受精后4小时（hours post-fertilization, hpf）至24hpf阶段，将斑马鱼胚胎暴露于两种不同浓度的乙醇溶液（1%与1.5%）中。miRNA表达谱分析结果显示，乙醇暴露可显著诱导miRNA表达失调。经两种浓度乙醇处理后，共有7种miRNA呈现普遍上调趋势，分别为miR-153a、miR-725、miR-30d、let-7k、miR-100、miR-738与miR-732；而仅在1%乙醇暴露组中，可检测到miR-23a、miR-203、let-7c、miR-128及miR-193b的表达下调。对失调miRNA的靶标预测结果表明，其推定靶标主要参与细胞周期调控、细胞凋亡及转录过程，而这些正是乙醇毒性所影响的核心生理过程。本研究整体结果显示，乙醇对miRNA表达失调的影响呈剂量依赖性，且miRNA在酒精毒性进程中发挥了相关作用。此外，本研究还获得了针对胚胎乙醇暴露的miRNA毒性特征谱。实验整体设计如下：斑马鱼胚胎由约10尾雄性与10尾雌性成年斑马鱼交配获得。收集胚胎后，将约250枚卵置于培养皿中，于28℃下孵育至4hpf（囊胚期）。在此阶段，通过体式显微镜观察胚胎发育情况，选取发育正常的胚胎（约200枚）用于乙醇暴露实验。具体操作如下：将200枚胚胎随机分配至装有20mL乙醇测试液的塑料培养皿中，测试液浓度分别为1%乙醇与1.5%乙醇，所有溶液均通过无水乙醇与系统水稀释配制。暴露处理时长为4hpf至24hpf。处理结束后，更换为系统水，继续培养胚胎至24hpf。对照组胚胎则仅培养于普通系统水中。于24hpf阶段收集斑马鱼胚胎，用于微阵列（microarray）分析。每个实验组均设置2次生物学重复。