microRNAs in Developmental Toxicology

描述(申请人提供)：先天畸形是人类发病率和死亡率的主要来源。化学物质暴露和其他环境因素在先天性畸形病因中的作用尚不完全清楚。然而，已知许多外来化学物质是实验动物的发育毒物或致畸物质，一些人类出生缺陷与胚胎暴露于外来生物物质有关。尽管有这样的理解，但大多数发育毒物和致畸剂扰乱胚胎发育的机制尚不清楚。在这次R21(探索性/发展研究)拨款申请中，我们建议研究一种新的潜在的发育毒性机制--干扰microRNA的表达--并建立斑马鱼胚胎作为研究microRNA在发育毒理学和致畸性中的作用的模型。MicroRNAs是一种~22个核苷酸的单链RNA分子，通过抑制mRNAs翻译成蛋白质并促进其隔离或降解来调节mRNAs的表达。MicroRNAs在胚胎发育过程中起着至关重要的作用；胚胎中microRNA表达或功能的中断可能会导致涉及多种组织的发育异常。在成年动物中，microRNAs也参与调节细胞对外来化学物质的反应。综上所述，这些结果表明，microRNAs表达的改变可能是发育毒物和致畸剂的一些影响的基础。然而，microRNAs在发育毒理学和畸形学中的可能作用尚未被探索。斑马鱼胚胎在理解微RNA在脊椎动物胚胎发育中的作用方面取得了一些根本性的进展。因此，斑马鱼胚胎是测试microRNAs表达变化是否是发育毒性的重要机制的一个很好的模型。在目标1中，我们将检验众所周知的发育毒物2，3，7，8-四氯二苯并对二恶英(TCDD)在斑马鱼胚胎中的发育毒性伴随着microRNAs表达的变化的假设。我们将比较两种microRNA表达谱分析方法：使用锁定核酸(LNA)探针的microRNA微阵列和深度测序(焦测序/454技术)。在目标2中，我们将测试这一假设，即在暴露于通过不同机制发挥作用的发育毒物后，会发生不同模式的microRNA表达变化。我们将根据目标1的结果选择一种microRNA图谱方法，并对暴露于已知人类致畸物质乙醇、丙戊酸和维甲酸的斑马鱼胚胎进行microRNA图谱分析。这些探索性实验将利用有关microRNA依赖的发育机制的新信息和新的深度测序技术来提供数据，这些数据可能会改变我们对发育毒理学所涉及机制的理解。公共卫生相关性：先天性畸形(出生缺陷)是人类发病率和死亡率的主要来源。化学物质暴露和其他环境因素在导致先天性畸形中的作用尚不完全清楚。然而，众所周知，许多药物和环境化学品是实验动物的发育毒物或致畸物质，一些人类出生缺陷与胚胎接触这些化合物密切相关。尽管有这样的理解，但大多数发育毒物和致畸物质扰乱胚胎发育的机制尚未完全阐明。在这里，我们建议研究发育毒性的新的潜在机制-干扰microRNA的表达。MicroRNAs是一种约22个核苷酸的单链RNA分子，通过抑制信使RNA翻译成蛋白质并促进其降解来调节信使RNA的表达。最近的研究已经证实，许多microRNAs在胚胎发育过程中发挥着关键作用。我们推测，某些发育毒物和致畸剂可能通过破坏microRNA的表达而起作用。这项拟议的研究将建立斑马鱼胚胎作为研究microRNAs在发育毒理学中的作用的有价值的模型。

DESCRIPTION (provided by applicant): Congenital malformations are a major source of human morbidity and mortality. The role of chemical exposure and other environmental factors in the etiology of congenital malformations is not completely understood. However, many xenobiotic chemicals are known to be developmental toxicants or teratogens in experimental animals and several human birth defects are associated with embryonic exposure to xenobiotics. Despite this understanding, the mechanisms by which most developmental toxicants and teratogens disrupt embryonic development are not known. In this R21 (Exploratory/Developmental Research) grant application, we propose to investigate a new potential mechanism of developmental toxicity-disruption of microRNA expression-and to establish the zebrafish embryo as a model for studying the roles of microRNAs in developmental toxicology and teratogenicity. MicroRNAs are single-stranded RNA molecules of ~22 nucleotides that regulate the expression of mRNAs by inhibiting their translation into proteins and promoting their sequestration or degradation. MicroRNAs have essential roles during embryonic development; disruption of microRNA expression or function in embryos can cause developmental abnormalities involving a variety of tissues. In adult animals, microRNAs also are involved in regulating cellular responses to xenobiotic chemicals. Together, these results suggest that altered expression of microRNAs might underlie some effects of developmental toxicants and teratogens. However, the possible role of microRNAs in developmental toxicology and teratology has not yet been explored. Zebrafish embryos have been instrumental in some of the fundamental advances in understanding the roles of microRNAs in embryonic development in vertebrates. Thus, the zebrafish embryo is an excellent model with which to test whether altered expression of microRNAs is an important mechanism of developmental toxicity. In aim 1, we will test the hypothesis that the developmental toxicity of the well-known developmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish embryos is accompanied by altered expression of microRNAs. We will compare two methods for microRNA expression profiling: microRNA microarrays using locked nucleic acid (LNA) probes, and deep sequencing (pyrosequencing / 454 technology). In aim 2, we will test the hypothesis that distinct patterns of altered microRNA expression occur after exposure to developmental toxicants that act via different mechanisms. We will choose one microRNA profiling method based on results of Aim 1 and perform microRNA profiling in zebrafish embryos exposed to the known human teratogens ethanol, valproic acid, and retinoic acid. These exploratory experiments will take advantage of new information on microRNA-dependent mechanisms of development and new deep sequencing technologies to provide data that could transform our understanding of mechanisms involved in developmental toxicology. PUBLIC HEALTH RELEVANCE: Congenital malformations (birth defects) are a major source of human morbidity and mortality. The role of chemical exposure and other environmental factors in contributing to congenital malformations is not completely understood. However, it is known that many drugs and environmental chemicals are developmental toxicants or teratogens in experimental animals and that several human birth defects are strongly associated with embryonic exposure to these compounds. Despite this understanding, the mechanisms by which most developmental toxicants and teratogens disrupt embryonic development have not yet been fully elucidated. Here we propose to investigate new potential mechanism of developmental toxicity- disruption of microRNA expression. MicroRNAs are single-stranded RNA molecules of ~22 nucleotides that regulate the expression of messenger RNAs by inhibiting their translation into proteins and promoting their degradation. Recent research has established that many microRNAs have critical roles during embryonic development. We hypothesize that some developmental toxicants and teratogens may act by disrupting microRNA expression. The proposed research will establish the zebrafish embryo as a valuable model for studying the roles of microRNAs in developmental toxicology.