Let-7 miRNA control of heart development

描述（由申请人提供）： 该申请涉及广泛的挑战领域（14）干细胞和特定的挑战主题，15-HL-102：基于microRNA技术开发心脏，肺和血液疾病的新治疗策略。先天性心脏畸形是最常见的出生缺陷。TBX 5是T-box转录因子家族的一个成员，其突变导致Holt-Oram综合征（HOS），这是一种以心脏和上肢畸形为特征的遗传性疾病。心脏对T-box基因剂量非常敏感，TBX 5表达的小扰动具有显著的心脏效应。microRNA（miRNAs）是一类长度约为22个核苷酸的内源性RNA，通过与靶基因的3 'UTR结合，在转录后水平调控基因的表达。它们微调靶基因的表达并调节许多生物过程，包括心脏发育。我们的初步数据表明let-7 miRNA可以通过结合TBX 5 3 'UTR中高度保守的靶位点来调控TBX 5的表达。我们已经鉴定了let-7成熟miRNA在鸡和小鼠胚胎心脏以及鸡前心外膜器官（PEO）中的表达。此外，我们已经表明，在鸡培养的心肌细胞中抑制let-7导致Nppa和Gja 5表达增加，这是TBX 5活性增加的标志。基于这些数据，我们假设let-7通过调节心肌和PEO中TBX 5的表达在心脏发生中起关键作用。因此，我们提出：（1）确定脊椎动物心脏发育过程中成熟let-7心脏表达的时空模式;（2）确定let-7修饰对心肌和心外膜器官中TBX 5功能的影响;（3）确定let-7敲低对哺乳动物心脏发生的影响。为了实现这些目标，我们将：（1）在鸡和小鼠心脏发育过程中进行let-7原位杂交，并采用“传感器”转基因研究let-7在小鼠心脏发生过程中的表达和活性;（2）在鸡中使用体细胞转基因以在心脏和PEO中过表达和下调let-7，并将测量细胞增殖和Nppa和Gja 5表达作为TBX 5活性的标志物，和（3）在基因工程小鼠模型中采用基于Cre驱动子的系统以谱系依赖性方式条件性下调let-7。这些实验将揭示心脏发育的基本途径，这可能有助于人类疾病的发病机制。我们将开发let-7表达的图谱和一系列腺病毒和小鼠试剂，这些试剂将成为社区在发育和成人背景下调查miRNAs的资源。由于miRNAs是未来治疗中最令人兴奋的靶分子之一，因此let-7/TBX 5调控网络的阐述有可能对先天性心脏病治疗策略的发展产生重大影响。 公共卫生相关性：这项研究致力于破译影响心脏的出生缺陷的分子机制。每年大约有4000名美国人死于这种心脏缺陷，识别调节导致这些疾病的分子事件的特定分子将为这些人提供新的治疗方法。该项目中正在研究的分子类型是治疗先天性心脏病患者的新方法的理想候选靶点。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (14) Stem Cells and specific Challenge Topic, 15-HL-102: Develop new therapeutic strategies for heart, lung, and blood diseases based on microRNA technology. Congenital heart malformations are the most common birth defects. Mutations in TBX5, a member of the T-box transcription factor family, cause Holt-Oram syndrome (HOS), a heritable disorder characterized by cardiac and upper limb malformations. The heart is exquisitely sensitive to T-box gene dosage, and small perturbations in TBX5 expression have significant cardiac effects. MicroRNAs (miRNAs) are endogenous ~22 nucleotides RNAs that regulate gene expression at the post-transcriptional level by binding the 3'UTR of target genes. They fine tune target genes' expression and regulate many biological processes, including heart development. Our preliminary data show that let-7 miRNA can regulate TBX5 expression by binding to a highly conserved target site in the TBX5 3'UTR. We have identified let-7 mature miRNA expression in both chick and mouse embryonic hearts and in the chick proepicardial organ (PEO). Moreover, we have shown that inhibition of let-7 in chick cultured cardiomyocytes leads to increased Nppa and Gja5 expression, a signature of increased TBX5 activity. Based on these data, we hypothesize that let-7 plays a critical role in cardiogenesis through the regulation of TBX5 expression in both the myocardium and the PEO. Thus, we propose (1) To define spatiotemporal patterns of mature let-7 cardiac expression during vertebrate heart development; (2) To identify the effect of let-7 modifications on TBX5 function in both the myocardium and the proepicardial organ, and (3) To determine the consequences of let-7 knockdown on mammalian cardiogenesis. To achieve these aims we will: (1) perform let-7 in situ hybridization during chick and mouse heart development and employ a "sensor" transgene to study let-7 expression and activity during mouse cardiogenesis; (2) use somatic transgenesis in chick to overexpress and downregulate let-7 in the heart and PEO and will measure cell proliferation and Nppa and Gja5 expression as markers of TBX5 activity, and (3) employ a Cre driver-based system to conditionally downregulate let-7 in a lineage dependent fashion in a genetically engineered mouse model. These experiments will shed light on a fundamental pathway in heart development that can contribute to the pathogenesis of human disease. We will develop an atlas of let-7 expression and a series of adenoviral and mouse reagents that will be resources for the community's investigation of miRNAs in both developmental and adult contexts. Since miRNAs are amongst the most exciting target molecules of future therapies, elaboration of a let-7/TBX5 regulatory network has the potential to generate major impact on the development of therapeutic strategies for congenital heart disease. Public Health Relevance: This research is devoted to deciphering molecular mechanisms that go awry in birth defects that affect the heart. Approximately 4000 Americans die annually with such heart defects, and identifying specific molecules that regulate the molecular events that cause these disorders will suggest new therapies for these individuals. The types of molecules being studied in this project are ideal candidate targets for such novel approaches to treat individuals with congenital heart defects.