LncRNA mechanism of heart failure

项目摘要/摘要 病理性应激诱导心肌转录基因重新编程，导致 心脏肌病和心力衰竭。这种应激诱导的基因重新编程需要在 染色质水平受染色质调节因子的影响。染色质调节剂的活性可以是 由一类新兴的RNA调制--长非编码RNA(LncRNAs)，它是 长RNA 具有低/无蛋白质编码潜力的转录本。LncRNA在肥厚和心力衰竭中的作用， 然而，基本上是未知的。大多数对lncRNA的研究是在细胞培养或酵母中进行的， 而老鼠的遗传模型也是缺乏的。本节目将重点研究一种新的小鼠遗传模型 确定一种新发现的LncRNA在控制心脏基因表达方面的分子功能， 肥大和失败。因为RNA可以被化学修饰并作为药物输送到 该方案的成功将为设计新的治疗机制奠定基础 治疗心力衰竭。 目的1：明确在心脏中对lncRNA的体内调控。我们将使用转基因和基因敲除 小鼠遗传学技术，以确定这种lncRNA的遗传和分子机制 表达在心脏中受到调节。方法还包括免疫染色、免疫印迹、定量 PCR、染色质免疫沉淀(ChIP)和RNA免疫沉淀(RNA-IP)分析。 目的2：确定该lncRNA如何与心脏中的染色质调节因子相互作用。我们会 使用分子、细胞和生物化学方法来确定其分子机制 LncRNA控制着心脏中染色质调节剂的分子功能。这些方法 包括RNA-IP、芯片、定量PCR、电迁移率改变分析、基于Episome报告 化验和核小体组装。 目的3：明确人lncRNA-蛋白质复合体的功能。我们将使用人类心脏组织和 IPS来源的心肌细胞证明存在LncRNA复合体。另外，我们将使用分子， 用细胞和生物化学方法定义人类lncRNA复合体的功能。这些方法 包括RNA-IP、少量组织芯片分析、定量聚合酶链式反应、电子迁移率漂移 分析、基于Episome的记者分析和基于iPS的技术。

Project Summary/Abstract Pathological stress induces transcriptional gene reprogramming in the heart muscle, leading to cardiac myopathy and heart failure. Such stress-induced gene reprogramming requires regulation at the chromatin level by chromatin-regulating factors. The activity of chromatin regulators can be modulated by an emerging class of RNAs-the long noncoding RNAs (lncRNAs), which are long RNA transcripts with low/no protein-coding potential. The role of lncRNAs in hypertrophy and heart failure, however, is essentially unknown. Most studies of lncRNAs are conducted in cell cultures or in yeast, and mouse genetic models are lacking. This program will focus on a new mouse genetic model to define the molecular function of a newly identified lncRNA in controlling cardiac gene expression, hypertrophy, and failure. Because RNAs can be chemically modified and delivered as a drug for therapy, the success of this program will lay down a foundation for designing new mechanism-based therapy for heart failure. Aim 1: Defining the in vivo regulation of an lncRNA in the heart. We will use transgenic and knockout technology of mouse genetics to define the genetic and molecular mechanisms by which this lncRNA expression is regulated in the heart. Methods also include immunostaining, western blot, quantitative PCR, chromatin immunoprecipitation (ChIP), and RNA immunoprecipitation (RNA-IP) analyses. Aim 2: Determining how this lncRNA interacts with chromatin-regulating factor in the heart. We will use molecular, cellular, and biochemical methods to determine the molecular mechanism by which the lncRNA controls the molecular functions of chromatin regulators in the hearts. The methods include RNA-IP, ChIP, quantitative PCR, electric mobility shift assays, episome-based reporter assays, and nucleosome assembly. Aim 3: Defining the function of human lncRNA-protein complex. We will use human heart tissues and iPS-derived heart cells to demonstrate the presence of lncRNA complex. Also, we will use molecular, cellular, and biochemical methods to define the function of human lncRNA complex. The methods include RNA-IP, ChIP analysis of small amount of tissues, quantitative PCR, electric mobility shift assays, episome-based reporter assays, and iPS-based technology.