Control of Cardiogenesis by microRNA Editing

描述(由申请人提供)： 这项应用解决了广泛的挑战领域(15)：翻译科学和特殊挑战主题15-HL-102：开发基于microRNA技术的心、肺和血液疾病的治疗策略。一种类型的RNA编辑涉及通过ADAR(作用于RNA的腺苷脱氨酶)的作用将双链RNA中的腺苷残基转化为肌苷。A？i RNA编辑对重要的哺乳动物基因如谷氨酸受体亚单位和5-羟色胺受体2C的功能进行重新编码和多样化。然而，A？I编辑最常见的目标是包含重复元件的反向重复的非编码RNA，例如位于内含子和3‘UTRs内的AlU和LINE。非编码、重复的RNA编辑的生物学意义在很大程度上是未知的。最近，我们发现某些miRNA基因的初级转录本(pri-miRNAs)被编辑。编辑pri-miRNAs会抑制它们对已编辑的成熟miRNAs的处理或表达，从而使不同于未编辑的miRNAs靶向的基因沉默。我们的发现揭示了A？I RNA编辑在miRNA介导的基因沉默中所起的先前未知的作用。心脏是胚胎组织，在E10期首次检测到ADAR1。ADAR2在胚胎10.5天的主动脉囊和胚胎14.5天的包括主动脉瓣在内的特定胚胎心脏区域均有表达。ADAR1缺失突变小鼠具有胚胎致死表型，包括心脏畸形，可能是由于心肌细胞增殖不足或凋亡增加所致。ADAR1和ADAR2编辑的未知目标dsRNA(S)可能在胚胎心脏发育的调节中发挥关键作用。MiRNA介导的RNA干扰最近作为一种以前未被怀疑的机制在发育过程中调节心脏发生。一组精选的miRNAs已被证明在胚胎心脏发育过程中抑制调控心肌细胞增殖/分化的基因。此外，这些miRNAs的异常表达与先天性室间隔缺陷或成人心脏的病理状况，如心室肥大、肥大和心律失常有关。我们的初步研究表明，miRNA-1、miRNA-133a和miR-208基因的初级转录本经历了A？I编辑。在这项挑战拨款申请中，我们将调查这些miRNAs的初级转录本的RNA编辑是否在调节心肌细胞增殖/分化和胚胎心脏发育中发挥作用。RNA编辑的效果将在体外通过miRNA处理实验和在体外诱导P19CL6小鼠胚胎干细胞向心肌细胞分化的过程中进行研究。这项建议中获得的信息将对基于miRNA的各种心血管疾病治疗的未来发展至关重要。 公共卫生相关性：我们的研究方案基于以前未探索的范例，将揭示关键信息，以更好地了解心脏正常和缺陷发育的潜在机制，以及成人心脏的功能和疾病。通过这项研究计划获得的信息对于未来基于miRNA的新干预措施的开发至关重要，该干预措施用于预防先天性心脏缺陷和病理性心脏疾病，如心肌肥大和心律失常。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Areas (15): Translational Science and Specific Challenge Topic 15-HL-102: Develop therapeutic strategies for heart, lung, and blood diseases based on microRNA technology. One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA by the action of ADAR (adenosine deaminase acting on RNA). The A?I RNA editing recodes and diversifies the function of important mammalian genes such as glutamate receptor subunits and serotonin receptor 2C. However, the most common targets for A?I editing are non-coding RNAs that contain inverted repeats of repetitive elements such as Alu and LINE located within introns and 3'UTRs. The biological significance of non-coding, repetitive RNA editing is largely unknown. Recently, we found that primary transcripts of certain miRNA genes (pri-miRNAs) are edited. Editing of pri-miRNAs results in inhibition of their processing or expression of edited mature miRNAs that silence genes different from those targeted by unedited miRNAs. Our findings revealed a previously unknown role for A?I RNA editing in miRNA-mediated gene silencing. The heart is the embryonic tissue where ADAR1 is first detected, at stage E10. ADAR2 expression is detected in the aortic sac at E10.5 and selected regions of embryonic heart including aortic valve at E14.5. ADAR1 null mutant mice have embryonic lethal phenotypes including heart malformation possibly due to hypoproliferation or increased apoptosis of cardiomyocytes. Editing of an unknown target dsRNA(s) by ADAR1 and ADAR2 may play a critical role in the regulation of embryonic heart development. The miRNA-mediated RNA interference recently emerged as a previously unsuspected mechanism to regulate cardiogenesis during development. A select set of miRNAs have been shown to repress genes that regulate proliferation/differentiation of cardiomyocytes during development of embryonic heart. Furthermore, aberrant expression of these miRNAs is associated with congenital ventricular septal defects or pathological conditions of adult heart such as ventricular hypertrophy, hyperplasia and arrhythmias. Our preliminary studies indicate that primary transcripts of miRNA-1, miRNA-133a and miR-208 genes undergo A?I editing. In this Challenge Grant application, we will investigate whether RNA editing of primary transcripts of these miRNAs play a role in the regulation of cardiomyocyte proliferation/differentiation and embryonic heart development. The effects of the RNA editing will be investigated in vitro by a miRNA processing assay and during in vitro induced differentiation of P19CL6 mouse embryonic stem cells to cardiomyocytes. Information obtained in this proposal will be essential for the future development of miRNA-based therapy of various cardiovascular diseases. Public Health Relevance: Our research proposal, based on previously unexplored paradigms, will reveal critical information for better understanding of the mechanisms underlying normal and defective development of heart, and function and disease of adult heart. Information obtained through this research proposal is essential for the future development of a new miRNA-based intervention for prevention of congenital heart defects and pathological cardiac conditions such as cardiac hypertrophy and arrhythmias.