Delta like-4 long non-coding RNA function in angiogenesis and vascular anomalies

Delta like-4长非编码RNA在血管生成和血管异常中的功能

• 批准号: 8919597
• 负责人: Ramani Ramchandran
• 金额: $ 49.24万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8919597
• 关键词: Adrenergic beta-Antagonists; Affect; Arteries; Binding; Blood Vessels; Cell Differentiation process; Cell Line; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Cellular biology; Chromatin; Clinic; Clinical; Code; Data; Development; Developmental Biology; Diagnostic; Disease; Embryo; Endothelial Cells; Event; Gene Expression Regulation; Gene Proteins; Generations; Genes; Genetic Transcription; Genome; Genomic Segment; Genomics; Goals; Growth; Growth Factor Inhibition; Health; Hemangioendothelioma; Hemangioma; Human; In Vitro; Indium; Intercistronic Region; Introns; Knowledge; Lung; Mediating; Messenger RNA; Mission; Modeling; Molecular; Mus; Pathogenesis; Patients; Peripheral arterial disease; Pharmaceutical Preparations; Play; Propranolol; Proteins; Public Health; RNA; RNA Interference; Regulation; Research; Retina; Role; Sampling; Signal Transduction; Slice; Testing; Therapeutic; Tissues; Transcript; Transcriptional Regulation; Tumor Angiogenesis; Untranslated RNA; Vascular Endothelial Growth Factors; Veins; Work; Zebrafish; angiogenesis; aptamer; base; cell type; histone modification; inhibitor/antagonist; innovation; knock-down; malformation; new growth; notch protein; novel; outcome forecast; promoter; public health relevance; response; secretase; therapeutic target; tool; transcription factor; tumor growth

 DESCRIPTION (provided by applicant): Our knowledge of coding RNAs, such as the function of mRNAs, has contributed immensely to our understanding of fundamental cellular processes such as regulation of gene expression and cell differentiation. Recently, transcripts in intergenic regions or within introns of vascular-specific genes are emerging as a new class of RNA molecules that may play a role in the intricate regulation of angiogenesis, the growth of new blood vessels from existing vasculature. These RNAs are referred to as non-coding RNAs (ncRNAs), and are classified as long (>200 bp) (lncRNAs) or short (<200 bp) (sncRNAs) depending on their sizes. Recent evidence suggests that a majority of lncRNAs in the genome do not code for proteins. They are located in the sense (S) or antisense (AS) orientation and, to date, the functional significance of these ncRNAs is poorly understood. Our long-term goal is to understand the underlying mechanisms utilized by ncRNAs during embryonic vascular development in order to effectively block them in disease states affected by deregulated vessel growth such as tumor angiogenesis and Vascular Anomalies (VAs). To pursue this long-term goal, the objective of this application is to study lncRNAs identified by our group for vascular gene delta like 4 (Dll4) that is located in AS direction to the Dll4 gene, and hereafter referred t as "Dll4AS." We have identified multiple lncRNAs (Dll4AS1-3) for the vascular gene Dll4 in mice, and each Dll4AS RNA is expressed to varying levels in murine endothelial cell line (MS1) and primary human endothelial cells (ECs). Our central hypothesis is that, "Transcriptional regulation of Dll4 occurs via a chromatin-mediated mechanism whereby regions in the Dll4 genomic locus are responsible for Dll4AS and Dll4 expression. This regulation is critical for normal angiogenesis (tip vs. stalk cell specification), and is deregulated in abnormal angiogenesis (artery-vein malformation), events associated with Notch signaling." This hypothesis is formulated based on preliminary data from our group that changes in both Dll4 and Dll4AS mRNA is observed under various experimental modulations such as cellular confluence, Notch inhibition, growth factor, and drug treatments. Further, we have identified a specific genomic region in the Dll4 locus that regulates the expression of both Dll4AS and Dll4 sense RNA, and knocking down the Dll4AS RNAs by silencing RNA-based approach in vitro in mouse ECs showed lower Dll4 expression in mouse ECs, and increases proliferation. Also, levels of both Dll4 and Dll4AS vary in different VAs sub-types. The proposed hypothesis will be tested by pursuing three specific aims: 1) Define the factors and mechanism involved in the regulation of Dll4 gene and Dll4AS; 2) Determine the role of dll4AS-dll4mRNA regulation in embryonic angiogenesis; and 3) Determine the extent of DLL4AS-DLL4 mRNA regulation in VAs. In each of these aims, we will employ a variety of cell biology, molecular, and developmental biology approaches to unravel the mechanistic basis for regulation of Dll4AS and Dll4 sense RNA in the developing vasculature, and its implications in VAs. The approach is innovative because exploiting the sensitivity of this regulation would benefit strategies where modulating the cognate transcript (DLL4) up or down using lncRNAs would be beneficial therapeutically for clinical conditions where more (peripheral artery disease) or less (tumor growth) angiogenesis is recommended. The proposed research is significant because identifying lncRNA signatures in select VA patient samples may serve as a diagnostic tool to distinguish between the sub-sets of these anomalies, and thus help in the accurate prognosis and treatment options in the clinic for these patients. DLL4AS RNA in itself could be a target for VAs, which would facilitate RNA-based therapeutic approaches such as Aptamers that have been successful in the clinic setting.

 描述(由申请人提供)：我们对编码RNA的知识，如mRNAs的功能，极大地促进了我们对基本细胞过程的理解，如基因表达和细胞分化的调节。最近，基因间的记录 血管特异基因的区域或内含子正在作为一类新的RNA分子出现，它们可能在血管生成的复杂调控中发挥作用，即从现有的血管系统生长新的血管。这些RNA被称为非编码RNA(NcRNAs)，并根据其大小被分类为长(&gt；200bp)(LncRNAs)或短(&lt；200bp)(NcRNAs)。最近的证据表明，基因组中的大多数lncRNAs不编码蛋白质。它们位于正义(S)或反义(AS)方向，迄今为止，人们对这些ncRNAs的功能意义知之甚少。我们的长期目标是了解ncRNAs在胚胎血管发育过程中使用的潜在机制，以便有效地阻止它们在受血管生长失调影响的疾病状态下，如肿瘤血管生成和血管异常(VAS)。为了追求这一长期目标，本申请的目的是研究我们团队鉴定的血管基因Delta like 4(DLL4)的lncRNAs，它位于DLL4基因的AS方向，以下称为DLL4AS。我们已经在小鼠中发现了血管基因DLL4的多个LncRNAs(Dll4AS1-3)，每个Dll4ASRNA在小鼠内皮细胞系(MS1)和原代人内皮细胞(ECs)中都有不同程度的表达。我们的中心假设是，“DLL4的转录调控是通过染色质介导的机制发生的，DLL4基因组中的区域负责DLL4AS和DLL4的表达。这种调控对正常的血管生成(尖端与茎细胞规格)至关重要，在异常血管生成(动-静脉畸形)中被解除调控，这是与Notch信号相关的事件。”这一假说是基于我们小组的初步数据提出的，即在各种实验调节下，如细胞融合、Notch抑制、生长因子和药物治疗，观察到DLL4和DLL4AS mRNA的变化。此外，我们在DLL4基因座上发现了一个调控DLL4AS和DLL4正义RNA表达的特定基因组区域，在体外通过基于RNA的沉默方法在小鼠内皮细胞中敲除DLL4AS RNA可以降低DLL4在小鼠内皮细胞中的表达，并促进细胞增殖。此外，DLL4和DLL4A的水平在不同的VAS亚型中也有所不同。这一假说将通过三个特定的目标来验证：1)确定参与DLL4基因和DLL4AS调控的因素和机制；2)确定DLL4AS-DLL4mRNA调控在胚胎血管生成中的作用；以及3)确定DLL4AS-DLL4mRNA在VAS中的调控程度。在这些目标中，我们将使用各种细胞生物学、分子和发育生物学方法来揭示DLL4AS和DLL4正义RNA在发育中的血管系统中调节的机制基础，以及它在VAS中的意义。这种方法是创新的，因为利用这一调节的敏感性将有利于使用lncRNA上调或下调同源转录本(DLL4)的策略，对于推荐更多(外周动脉疾病)或更少(肿瘤生长)血管生成的临床情况将是有益的治疗。这项拟议的研究具有重要意义，因为在精选的VA患者样本中识别lncRNA特征可以作为一种诊断工具来区分这些异常的子集，从而有助于临床上对这些患者的准确预后和治疗选择。DLL4AS RNA本身可能是VAS的靶点，这将促进基于RNA的治疗方法，如已在临床上成功的适体。