Control of Cardiogenesis by microRNA Editing

描述（由申请人提供）： 该应用解决了广泛的挑战领域 (15)：转化科学和具体挑战主题 15-HL-102：基于 microRNA 技术制定心脏、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 编辑重新编码重要哺乳动物基因的功能并使其功能多样化，例如谷氨酸受体亚基和血清素受体 2C。然而，A?I 编辑最常见的目标是非编码 RNA，它们包含重复元件的反向重复，例如位于内含子和 3'UTR 内的 Alu 和 LINE。非编码、重复性 RNA 编辑的生物学意义在很大程度上尚不清楚。最近，我们发现某些 miRNA 基因 (pri-miRNA) 的初级转录本被编辑。 pri-miRNA 的编辑会抑制其加工或编辑成熟 miRNA 的表达，从而沉默与未编辑 miRNA 靶向的基因不同的基因。我们的研究结果揭示了 A?I RNA 编辑在 miRNA 介导的基因沉默中的先前未知的作用。心脏是 ADAR1 在 E10 阶段首次被检测到的胚胎组织。在 E10.5 时在主动脉囊中检测到 ADAR2 表达，在 E14.5 时在胚胎心脏的选定区域（包括主动脉瓣）中检测到 ADAR2 表达。 ADAR1 缺失突变小鼠具有胚胎致死表型，包括可能由于心肌细胞增殖不足或凋亡增加而导致的心脏畸形。 ADAR1 和 ADAR2 对未知靶标 dsRNA 的编辑可能在胚胎心脏发育的调节中发挥关键作用。 miRNA 介导的 RNA 干扰最近作为一种先前未被怀疑的机制出现，可以在发育过程中调节心脏发生。一组选定的 miRNA 已被证明可以抑制在胚胎心脏发育过程中调节心肌细胞增殖/分化的基因。此外，这些 miRNA 的异常表达与先天性室间隔缺损或成人心脏的病理状况（例如心室肥厚、增生和心律失常）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。在本次挑战资助申请中，我们将研究这些 miRNA 初级转录物的 RNA 编辑是否在心肌细胞增殖/分化和胚胎心脏发育的调节中发挥作用。将通过 miRNA 加工测定以及 P19CL6 小鼠胚胎干细胞体外诱导分化为心肌细胞的过程来研究 RNA 编辑的影响。该提案中获得的信息对于未来开发基于 miRNA 的各种心血管疾病疗法至关重要。 公共健康相关性：我们的研究提案基于先前未探索的范式，将揭示关键信息，以便更好地理解心脏正常和缺陷发育以及成人心脏功能和疾病的机制。通过这项研究提案获得的信息对于未来开发一种新的基于 miRNA 的干预措施至关重要，以预防先天性心脏缺陷和病理性心脏疾病，如心脏肥大和心律失常。

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.