microRNA and Down Syndrome

描述(申请人提供)：唐氏综合征(DS)的染色体异常是由人类21号染色体(Hsa21)的一部分三倍体引起的，但这种染色体异常如何导致DS表型尚不清楚。目前的提案将直接解决这一问题，重点是一类新的内源性基因调节因子，microRNAs(MiRNAs)。MiRNAs通常被认为是基因表达的负调控因子，通过与蛋白质编码的mRNA转录本中的互补序列进行碱基配对来抑制翻译和/或促进信使RNA(MRNA)的降解。我们最近的生物信息学分析证实，Hsa21含有5个miRNA基因。重要的是，miRNA表达谱、miRNA RT-PCR和miRNA原位杂交实验表明，所有五个源于Hsa21的miRNAs在DS患者的脑和心脏标本中都过度表达。我们现在假设，五个Hsa21衍生的miRNAs的过度表达导致了一些重要的蛋白质靶标的低表达，这些蛋白质靶标在一定程度上导致了DS的表型。生物信息学分析表明，数千种蛋白质可能受这些miRNAs的调控。由于联合靶向多个miRNAs和单个mRNA可能导致比少数miRNAs靶向的mRNAs更明显的下调，因此重新分析了所有Hsa21衍生的miRNA/mRNA对是否存在多个Hsa21衍生的miRNA结合位点。随后，根据单个靶基因在DS中发挥作用的潜在临床相关性，对候选靶点列表进行了优先排序。基于这些标准，我们选择研究甲基CpG结合蛋白(MeCP2)，一种潜在的重要的Hsa21来源的miRNA靶标，因为它的34个非翻译区包含至少一个假定的识别位点，所有Has21来源的miRNAs。此外，MeCP2是一个具有挑衅性的miRNA靶标，因为该基因的突变会导致Rett综合征，这是一种神经发育障碍，与DS中观察到的一些神经异常相似。我们的初步数据现在表明，MeCP2 mRNA是Hsa21来源的miR-155的直接靶点，并且MeCP2在人胎儿和成人DS脑标本以及DS小鼠模型中表达不足。作为MeCP2表达减弱的结果，转录激活和沉默的MeCP2靶基因在这些DS脑标本中被异常调控。为了证实Hsa21衍生的miRNAs在DS中的因果作用，通过脑室内注射antagomir-155在体内沉默内源性成熟miR-155的表达，导致DS小鼠脑中miR-155和MeCP2的表达水平正常化。综上所述，这些初步数据表明，MeCP2的不当抑制继发于miR-155三体的过度表达，导致MeCP2靶基因的异常调节。这种失调随后会导致重要的“调节回路”的不稳定，这在一定程度上导致了DS患者的认知缺陷。公共卫生相关性：这个项目代表了一条关于DS分子机制的新的研究路线。这项研究将为Hsa21来源的miRNAs抑制关键调控蛋白的表达提供“概念证据”，这反过来又导致一些对神经发育至关重要的因子的异常表达。我们的方法包括综合和多学科的方法，并包括人体组织、细胞系和DS小鼠模型。我们的项目将确定与DS有关的miRNA/mRNA靶点，并可能导致在围产期治疗DS患者的新治疗策略，以改变发病过程。

DESCRIPTION (provided by applicant): The chromosome abnormality in Down syndrome (DS) results from a triplication in a portion of human chromosome 21 (Hsa21), but how this chromosomal anomaly causes the DS phenotype is not clear. The current proposal will directly address this issue, with an emphasis on a novel class of endogenous gene regulators, microRNAs (miRNAs). MiRNAs are generally regarded as negative regulators of gene expression that inhibit translation and/or promote messenger RNA (mRNA) degradation by base-pairing to complementary sequences within protein-coding mRNA transcripts. Our recent bioinformatic analyses established that Hsa21 harbors five miRNA genes. Importantly, miRNA expression profiling, miRNA RT-PCR, and miRNA in situ hybridization experiments demonstrated that all five Hsa21-derived miRNAs are over-expressed in brain and heart specimens from individuals with DS. We now hypothesize that the over-expression of the five Hsa21-derived miRNAs results in the under-expression of a number of important protein targets which contribute, in part, to the DS phenotype. Bioinformatic analyses demonstrated that several thousand proteins may be regulated by these miRNAs. Because combinatorial targeting of multiple miRNAs with a single mRNA may lead to a more pronounced down-regulation relative to mRNAs targeted by a few miRNAs, all of the Hsa21-derived miRNA/mRNA pairs were re-analyzed for the presence of multiple Hsa21-derived miRNA binding sites. This list of candidate targets was subsequently prioritized with respect to the potential clinical relevance of an individual target gene in playing a role in DS. Based on these criteria, we chose to investigate the methyl-CpG-binding protein (MeCP2), a transcription factor, as a potentially important Hsa21- derived miRNA target since its 34-untranslated region harbors at least one putative recognition site for all of the Has21-derived miRNAs. Additionally, MeCP2 is a provocative miRNA target since mutations in this gene contribute to Rett syndrome, a neurodevelopmental disorder that shares some of the neurologic abnormalities observed in DS. Our preliminary data now demonstrate that MeCP2 mRNA is a direct target of Hsa21-derived miR-155 and that MeCP2 is under-expressed in human fetal and adult DS brain specimens and in a mouse model of DS. As a consequence of attenuated MeCP2 expression, transcriptionally-activated and -silenced MeCP2 target genes are aberrantly regulated in these DS brain specimens. To begin to substantiate a causal role of Hsa21-derived miRNAs in DS, in vivo silencing of endogenous mature miR-155 expression by intra- ventricular injection of antagomir-155 resulted in the normalization of miR-155 and MeCP2 expression levels in the DS mouse brains. Taken together, these preliminary data suggest that improper repression of MeCP2, secondary to trisomic over-expression of miR-155, result in the aberrant regulation of MeCP2 target genes. This dysregulation subsequently results in the destabilization of important "regulatory circuits" that contribute, in part, to the cognitive defects that occur in DS individuals. PUBLIC HEALTH RELEVANCE: This project represents a novel line of inquiry regarding the molecular mechanisms of DS. This study will provide "proof of concept" that Hsa21-derived miRNAs inhibit the expression of critical regulatory proteins, which in turn, results in aberrant expression of a number of factors critical for neurodevelopment. Our approach includes a comprehensive and multi-disciplinary approach and includes human tissues, cell lines, and a DS mouse model. Our project will define miRNA/mRNA targets responsible for DS and will potentially lead to novel therapeutic strategies to treat DS individuals in the perinatal period to change the course of pathogenesis.