In-vivo Function and Mechanism of the Long non-coding RNA Locus Handsdown

长非编码RNA基因座的体内功能和机制

• 批准号: 403634508
• 负责人: Dr. Phillip Grote
• 金额: --
• 依托单位: Georg-Speyer-Haus, Institut für Tumorbiologie und experimentelle Therapie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/403634508?language=en
• 关键词: vivo; Function; Mechanism; Long; non

Heart malfunction is one of the major causes of morbidity and lethality in modern society and nearly 1 in 100 newborns exhibit developmental malformations of the heart. In addition, heart injuries such as myocardial infarction impose an enormous societal and economic burden as they often result in maladaptive heart remodeling leading to congestive heart failure. While the heart has very limited regenerative potential, numerous efforts are currently undertaken to enhance regeneration or transplant cells with regenerative potential to patients. However, an in-depth knowledge of how the heart develops under normal circumstances is crucial for the development of effective regenerative therapies for heart disease.The heart is the first organ to develop during embryogenesis, containing functional cells in the form of contractile cardiomyocytes from very early on. Shortly after birth, the heart of both mice and humans has the temporally-restricted potential to regenerate injured heart muscle tissue into fully functional contractile tissue. While the role of many protein-coding RNAs in heart development has been well-studied, the class of long non-coding RNAs (lncRNAs) and their role in heart development and disease has only recently shifted into focus. Actually, the most abundant RNA transcripts in the vertebrate transcriptome are long noncoding RNAs (lncRNAs), transcripts that are longer than 200 nucleotides and do not encode proteins. LncRNAs have been associated with epigenetic changes, developmental defects and cancer, and knockouts are sometimes lethal. We showed previously that the lncRNA Fendrr interacts with the Polycomb repressive complex 2 (PRC2) and thereby regulates essential genes in the early embryonic cardiac cell lineage. We identified several novel lncRNAs with specific expression in the early embryonic heart and show that one particular locus is essential for embryonic development. To that end, we deleted the lncRNA locus in mouse embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing and generated mouse embryos from these ESCs. The resulting embryos are embryonic lethal with, amongst other defects, mis-development of the heart. We propose here to analyze this novel heart specific lncRNA locus in vivo using state-of the art genomic tools and the generation of embryos and mice directly from genetically engineered stem cells. We will furthermore define the molecular mechanism of this lncRNA locus with respect to the RNA transcripts generated from it or the underlying genomic context.By understanding how lncRNAs function during normal heart development, we are likely to define new lncRNAs that may be valid targets to reactivate regenerative potential in adult hearts or enhance the regenerative potential of transplanted cells. Furthermore, successful downregulation of the lncRNA transcript by antisense RNA technology (GapmerR) might allow for the upregulation of heart specific genes, beneficial for cardiac repair after injury.

心脏功能障碍是现代社会发病和致死的主要原因之一，近1/100的新生儿表现出心脏发育畸形。此外，心脏损伤如心肌梗死造成巨大的社会和经济负担，因为它们经常导致适应不良的心脏重塑，导致充血性心力衰竭。虽然心脏具有非常有限的再生潜力，但目前正在进行许多努力以增强再生或将具有再生潜力的细胞移植给患者。然而，深入了解心脏在正常情况下如何发育，对于开发有效的心脏病再生疗法至关重要。心脏是胚胎发育过程中发育的第一个器官，从很早开始就含有收缩性心肌细胞形式的功能细胞。出生后不久，小鼠和人的心脏都具有暂时受限的将受损的心肌组织再生为完全功能性收缩组织的潜力。虽然许多蛋白质编码RNA在心脏发育中的作用已经得到了很好的研究，但长链非编码RNA（lncRNA）及其在心脏发育和疾病中的作用最近才成为焦点。事实上，脊椎动物转录组中最丰富的RNA转录物是长非编码RNA（lncRNA），即长于200个核苷酸且不编码蛋白质的转录物。lncRNA与表观遗传变化、发育缺陷和癌症有关，敲除有时是致命的。我们先前表明，lncRNA Fendrr与Polycomb抑制复合物2（PRC 2）相互作用，从而调节早期胚胎心脏细胞谱系中的必需基因。我们鉴定了几种在早期胚胎心脏中具有特异性表达的新型lncRNA，并表明一个特定的位点对胚胎发育至关重要。为此，我们使用CRISPR/Cas9基因组编辑删除了小鼠胚胎干细胞（ESC）中的lncRNA基因座，并从这些ESC中生成了小鼠胚胎。由此产生的胚胎是胚胎致命的，除其他缺陷外，还包括心脏发育不良。我们建议在这里分析这种新的心脏特异性lncRNA基因座在体内使用国家的最先进的基因组工具和胚胎和小鼠直接从基因工程干细胞的一代。我们将进一步定义这个lncRNA基因座的分子机制，从它或潜在的基因组context.By了解lncRNA如何在正常心脏发育过程中发挥作用，我们可能会定义新的lncRNA，可能是有效的目标，以重新激活再生潜力在成人心脏或增强移植细胞的再生潜力。此外，通过反义RNA技术（GapmerR）成功下调lncRNA转录物可能允许上调心脏特异性基因，这有利于损伤后的心脏修复。