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mRNA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控。最近，基因间转录本 区域内或血管特异性基因的内含子内的RNA分子正在作为一类新的RNA分子出现，其可能在血管生成的复杂调节中起作用，所述血管生成是从现有脉管系统生长新血管。这些RNA被称为非编码RNA（ncRNA），并根据其大小分为长（>200 bp）（lncRNA）或短（<200 bp）（sncRNA）。最近的证据表明，基因组中的大多数lncRNA不编码蛋白质。它们位于正义（S）或反义（AS）方向，迄今为止，这些ncRNA的功能意义知之甚少。我们的长期目标是了解ncRNA在胚胎血管发育过程中所利用的潜在机制，以便在受血管生长失调影响的疾病状态（如肿瘤血管生成和血管异常（VA））中有效地阻断它们。为了追求这一长期目标，本申请的目的是研究由我们的小组鉴定的位于Dll 4基因的AS方向的血管基因δ样4（Dll 4）的lncRNA，下文称为“Dll 4AS”。“我们已经在小鼠中鉴定了血管基因Dll 4的多种lncRNA（Dll 4AS 1 -3），并且每种Dll 4AS RNA在鼠内皮细胞系（MS 1）和原代人内皮细胞（EC）中表达不同的水平。我们的中心假设是，“Dll 4的转录调节通过染色质介导的机制发生，其中Dll 4基因组位点中的区域负责Dll 4AS和Dll 4表达。这种调节对于正常的血管生成（尖端细胞与柄细胞特化）是至关重要的，并且在异常血管生成（动脉-静脉畸形）（与Notch信号传导相关的事件）中是失调的。“这一假设是基于我们小组的初步数据制定的，即在各种实验调节下观察到Dll 4和Dll 4AS mRNA的变化，如细胞融合，Notch抑制，生长因子和药物治疗。此外，我们已经鉴定了D114基因座中调节D114 AS和D114正义RNA表达的特定基因组区域，并且在小鼠EC中通过基于RNA的沉默方法体外敲低D114 AS RNA显示小鼠EC中较低的D114表达，并且增加增殖。此外，Dll 4和Dll 4AS的水平在不同的VA亚型中不同。本研究将通过以下三个具体目标来验证这一假设：1）确定Dll 4基因和Dll 4AS调控的因素和机制; 2）确定dll 4AS-dll 4 mRNA调控在胚胎血管生成中的作用; 3）确定VA中DLL 4AS-DLL 4 mRNA调控的程度。在这些目标中的每一个，我们将采用各种细胞生物学，分子和发育生物学的方法来解开Dll 4AS和Dll 4正义RNA在发展中的血管系统中的调节的机制基础，及其在VA中的意义。该方法是创新性的，因为利用这种调节的敏感性将有益于其中使用lncRNA上调或下调同源转录物（DLL 4）的策略将在治疗上有益于推荐更多（外周动脉疾病）或更少（肿瘤生长）血管生成的临床病症。拟议的研究是重要的，因为在选定的VA患者样本中识别lncRNA签名可以作为一种诊断工具来区分这些异常的子集，从而有助于这些患者在临床上的准确预后和治疗选择。DLL 4AS RNA本身可能是VA的靶点，这将促进基于RNA的治疗方法，如在临床环境中取得成功的适体。