microRNAs in Developmental Toxicology

描述（由申请人提供）：先天性畸形是人类发病率和死亡率的主要来源。化学暴露和其他环境因素在先天性畸形病因学中的作用尚不完全清楚。然而，许多外来化学物质在实验动物中被认为是发育毒性物质或致畸物，一些人类出生缺陷与胚胎接触外来化学物质有关。尽管了解了这一点，但大多数发育毒性物质和致畸物破坏胚胎发育的机制尚不清楚。在本次R21（探索性/发展性研究）资助申请中，我们建议研究一种新的潜在的发育毒性机制- microRNA表达的破坏-并建立斑马鱼胚胎作为研究microRNA在发育毒理学和致畸中的作用的模型。microrna是约22个核苷酸的单链RNA分子，通过抑制mrna转化为蛋白质并促进其隔离或降解来调节mrna的表达。MicroRNAs在胚胎发育过程中发挥重要作用；胚胎中microRNA表达或功能的破坏可导致涉及多种组织的发育异常。在成年动物中，microrna也参与调节细胞对外来化学物质的反应。总之，这些结果表明，microrna表达的改变可能是发育毒物和致畸物的一些影响的基础。然而，microrna在发育毒理学和致畸学中的可能作用尚未被探索。斑马鱼胚胎在理解微小rna在脊椎动物胚胎发育中的作用方面取得了一些基本进展。因此，斑马鱼胚胎是一个很好的模型，用来测试microrna表达的改变是否是发育毒性的重要机制。在目标1中，我们将验证一个假设，即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microrna表达的改变。我们将比较microRNA表达谱的两种方法：使用锁定核酸（LNA）探针的microRNA微阵列和深度测序（焦磷酸测序/ 454技术）。在目标2中，我们将检验这样一种假设，即暴露于通过不同机制起作用的发育毒物后，microRNA表达的不同模式会发生改变。我们将根据Aim 1的结果选择一种microRNA分析方法，并在暴露于已知人类致畸物乙醇、丙戊酸和视黄酸的斑马鱼胚胎中进行microRNA分析。这些探索性实验将利用microrna依赖的发育机制和新的深度测序技术的新信息，提供可以改变我们对发育毒理学机制的理解的数据。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。化学接触和其他环境因素在导致先天畸形中的作用还不完全清楚。然而，众所周知，在实验动物中，许多药物和环境化学物质是发育毒性物质或致畸物，人类的一些出生缺陷与胚胎时期接触这些化合物密切相关。尽管有这样的认识，大多数发育毒性物质和致畸物破坏胚胎发育的机制尚未完全阐明。在此，我们提出研究一种新的潜在的发育毒性机制-破坏microRNA的表达。microrna是一种约22个核苷酸的单链RNA分子，通过抑制信使RNA转化为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microrna在胚胎发育过程中起着关键作用。我们假设一些发育毒性物质和致畸物可能通过破坏microRNA表达而起作用。本研究将建立斑马鱼胚胎作为研究microrna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.