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

 描述（由申请人提供）：我们对编码 RNA（例如 mRNA 的功能）的了解极大地促进了我们对基本细胞过程（例如基因表达调节和细胞分化）的理解。最近，基因间转录本 血管特异性基因的区域或内含子内正在作为一类新的RNA分子出现，它们可能在血管生成（现有脉管系统新血管的生长）的复杂调节中发挥作用。这些 RNA 被称为非编码 RNA (ncRNA)，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。它们位于有义 (S) 或反义 (AS) 方向，迄今为止，人们对这些 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利用的潜在机制，以便有效地阻止它们处于受血管生长失调影响的疾病状态，例如肿瘤血管生成和血管异常 (VA)。为了实现这一长期目标，本申请的目的是研究我们小组鉴定的血管基因δ样4（Dll4）的lncRNA，该基因位于Dll4基因的AS方向，以下简称“Dll4AS”。我们在小鼠体内鉴定出了多种与血管基因 Dll4 相关的 lncRNA (Dll4AS1-3)，并且每种 Dll4AS RNA 在小鼠内皮细胞系 (MS1) 和原代人内皮细胞 (EC) 中都有不同的表达水平。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，其中 Dll4 基因座中的区域负责 Dll4AS 和 Dll4 的表达。这种调控对于正常血管生成（尖端与茎细胞规范）至关重要，并且在与 Notch 信号传导相关的异常血管生成（动静脉畸形）中失调。”这一假设是根据我们小组的初步数据制定的，即在各种实验调节（例如细胞汇合、Notch 抑制、生长因子和药物治疗）下观察到 Dll4 和 Dll4AS mRNA 的变化。此外，我们还鉴定了 Dll4 基因座中的一个特定基因组区域，该区域调节 Dll4AS 和 Dll4 sense RNA 的表达，并且通过在体外小鼠 EC 中沉默基于 RNA 的方法敲低 Dll4AS RNA，显示小鼠 EC 中 Dll4 表达较低，并增加增殖。此外，不同 VA 亚型中 Dll4 和 Dll4AS 的水平也有所不同。所提出的假设将通过三个具体目标进行检验：1）明确Dll4基因和Dll4AS调控所涉及的因素和机制； 2）确定dll4AS-dll4mRNA调控在胚胎血管生成中的作用； 3) 确定 VA 中 DLL4AS-DLL4 mRNA 调节的程度。在每个目标中，我们将采用各种细胞生物学、分子和发育生物学方法来揭示发育中脉管系统中 Dll4AS 和 Dll4 sense RNA 调节的机制基础及其对 VA 的影响。该方法具有创新性，因为利用这种调节的敏感性将有利于使用lncRNA向上或向下调节同源转录本（DLL4）的策略，这对于建议增加（外周动脉疾病）或更少（肿瘤生长）血管生成的临床病症有治疗效果。拟议的研究意义重大，因为在选定的 VA 患者样本中识别 lncRNA 特征可以作为区分这些异常子集的诊断工具，从而有助于在临床上为这些患者提供准确的预后和治疗选择。 DLL4AS RNA 本身可以成为 VA 的靶标，这将促进基于 RNA 的治疗方法，例如在临床环境中取得成功的适配体。

 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.