LncRNA mechanism of heart failure

描述（由申请人提供）：病理性应激诱导心肌转录基因重编程，导致心肌病和心力衰竭。这种应激诱导的基因重编程需要染色质调节因子在染色质水平上进行调控。染色质调控因子的活性可以被一类新兴的RNA -长链非编码RNA （lncRNAs）调节，这是一种具有低或无蛋白质编码潜力的长链RNA转录物。然而，lncrna在肥厚和心力衰竭中的作用基本上是未知的。大多数lncrna的研究都是在细胞培养或酵母中进行的，缺乏小鼠遗传模型。本项目将重点研究一种新的小鼠遗传模型，以确定一种新发现的lncRNA在控制心脏基因表达、肥大和衰竭中的分子功能。由于rna可以被化学修饰并作为治疗药物传递，因此该项目的成功将为设计基于新机制的心力衰竭治疗奠定基础。目的1：确定心脏中lncRNA的体内调控。我们将利用小鼠遗传学的转基因和基因敲除技术来确定这种lncRNA在心脏中表达调控的遗传和分子机制。方法还包括免疫染色、western blot、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。我们将使用分子，细胞和生化方法来确定lncRNA控制心脏中染色质调节因子的分子功能的分子机制。方法包括RNA-IP、ChIP、定量PCR、电迁移转移测定、基于发作体的报告基因测定和核小体组装。目的3：明确人lncrna -蛋白复合物的功能。我们将使用人类心脏组织和ips衍生的心脏细胞来证明lncRNA复合物的存在。此外，我们将使用分子，细胞和生化方法来定义人类lncRNA复合物的功能。这些方法包括RNA-IP、少量组织的ChIP分析、定量PCR、电迁移转移分析、基于发作体的报告基因分析和基于ip的技术。

DESCRIPTION (provided by applicant): 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.