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: 9265498
• 负责人: Ramani Ramchandran
• 金额: $ 42.11万
• 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/9265498
• 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; Elements; Embryo; Endothelial Cells; Event; Gene Expression Regulation; Gene Proteins; Generations; Genes; Genetic Transcription; Genome; Genomic Segment; Genomics; Goals; Growth; Growth Factor; Health; Hemangioendothelioma; Hemangioma; Human; In Vitro; Intercistronic Region; Introns; Knowledge; Lung; Mediating; Messenger RNA; Mission; Modeling; Molecular Biology; Mus; Pathogenesis; Patients; Peripheral arterial disease; Pharmacotherapy; Play; Propranolol; Proteins; Public Health; RNA; RNA Interference; Regulation; Research; Retina; Role; Sampling; Signal Transduction; Slice; Testing; Therapeutic; Tissues; Transcript; Transcriptional Regulation; Tumor Angiogenesis; United States National Institutes of Health; Untranslated RNA; Vascular Endothelial Growth Factors; Veins; Zebrafish; angiogenesis; aptamer; base; cell type; gamma secretase; histone modification; inhibitor/antagonist; innovation; knock-down; malformation; new growth; notch protein; novel; outcome forecast; promoter; public health relevance; response; 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（应用程序提供）：我们对编码RNA的了解，例如mRNA的功能，为我们对基本细胞过程（例如调节基因表达和细胞分化的调节）的理解做出了巨大贡献。最近，基因间的成绩单 区域或血管特异性基因的介绍中正在成为一种新的RNA分子，这些分子可能在复杂的血管生成，即现有脉管系统中新血管的生长中发挥作用。这些RNA被称为非编码RNA（NCRNA），并将其分类为长（> 200 bp）（lncrNA）或短（<200 bp）（sncRNA）（SNCRNA），具体取决于它们的大小。最近的证据表明，基因组中的大多数LNCRNA不会为蛋白质编码。它们位于意义上（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使用的基本机制，以便在受肿瘤血管生成和血管异常（VAS）等血管生长（例如）影响的疾病状态下有效阻止它们。为了实现这一长期目标，该应用的目的是研究我们小组确定的血管基因delta识别的lncrnas，如4（dll4），它位于DLL4基因的方向上，并以下称为“ dll4as”。我们已经确定了小鼠血管基因DLL4的多个LNCRNA（DLL4AS1-3），并且每个DLL4AS RNA在鼠内皮细胞系（MS1）和原发性人体内皮细胞（EC）中表示为不同的水平。 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."该假设是根据我们组的初步数据提出的，即在各种实验调制（例如细胞汇合，缺口抑制，生长因子和药物处理）下观察到DLL4和DLL4AS mRNA的变化。此外，我们已经确定了DLL4基因座中的一个特定基因组区域，该区域调节了DLL4AS和DLL4 Sense RNA的表达，并通过在小鼠EC中基于RNA的体外方法来击倒DLL4AS RNA，显示出小鼠EC中的较低DLL4表达在小鼠ECS中的表达较低，并增加了扩散。同样，在不同的VAS子类型中，DLL4和DLL4A的水平都不同。提出的假设将通过追求三个具体目的来检验：1）定义调节DLL4基因和DLL4AS的因素和机制； 2）确定DLL4AS-DLL4MRNA调节在胚胎血管生成中的作用； 3）确定VAS中DLL4AS-DLL4MRNA调控的程度。在这些目标中的每一个中，我们将采用各种细胞生物学，分子和发育生物学方法来阐明发育中的脉管系统中DLL4AS和DLL4感应RNA调节的机械基础，及其在VAS中的含义。该方法具有创新性，因为利用该法规的敏感性将使使用LNCRNA上下调节同源转录本（DLL4）的策略有益于临床状况是有益的理论，即推荐更多（周围动脉疾病）或更少（肿瘤生长）血管生成。拟议的研究很重要，因为在某些VA患者样品中鉴定LNCRNA特征可能是区分这些异常的子集的诊断工具，因此有助于这些患者的诊所的准确预后和治疗选择。 DLL4AS RNA本身可能是VAS的目标，这将促进基于RNA的治疗方法，例如在临床环境中成功的apamers。