LncRNA mechanism of heart failure

LncRNA心力衰竭机制

• 批准号: 8612415
• 负责人: CHING-PIN CHANG
• 金额: $ 40.04万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8612415
• 关键词: Adult; Animals; Antisense RNA; Biochemical; Biological Assay; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Culture Techniques; Chemicals; Chromatin; Chromatin Remodeling Factor; Code; Complex; Data; Development; Electrophoretic Mobility Shift Assay; Epigenetic Process; Episome; FDA approved; Failure; Feedback; Fetal Heart; Fibrosis; Foundations; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Models; Heart; Heart Hypertrophy; Heart failure; Homeostasis; Human; Hypertrophy; Immunoprecipitation; Knock-out; Methods; Modeling; Molecular; Molecular Genetics; Morbidity - disease rate; Mus; Myocardium; Myopathy; Myosin Heavy Chains; Neonatal; Nucleosomes; Nucleotides; Outcome; Pathogenesis; Pharmacotherapy; Proteins; RNA; RNA Splicing; Regulation; Reporter; Repression; Role; SMARCA4 gene; Societies; Stress; Switch Genes; Technology; Tissues; Transcript; Transgenic Organisms; Untranslated RNA; Western Blotting; Work; Yeasts; arm; base; chromatin immunoprecipitation; chromatin remodeling; design; heart cell; heart function; human disease; in vivo; induced pluripotent stem cell; insight; mortality; pressure; prevent; programs; promoter; protein complex; public health relevance; restoration; success

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.

项目总结/摘要 病理性应激诱导心肌中的转录基因重编程，导致 心肌病和心力衰竭。这种压力诱导的基因重编程需要调节， 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 由一类新出现的RNA-长链非编码RNA（lncRNA）调节， 长RNA 具有低/无蛋白质编码潜力的转录物。lncRNA在肥大和心力衰竭中的作用， 然而，基本上是未知的。大多数lncRNA的研究是在细胞培养物或酵母中进行的， 缺乏小鼠遗传模型。该计划将专注于一种新的小鼠遗传模型， 确定了一种新鉴定的lncRNA在控制心脏基因表达中的分子功能， 肥大和衰竭。因为RNA可以被化学修饰并作为药物递送， 该项目的成功将为设计新的基于机制的治疗方法奠定基础。 治疗心力衰竭 目的1：确定心脏中lncRNA的体内调节。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、蛋白质印迹、定量免疫荧光、免疫荧光、免疫组织化学、免疫组织化学和免疫组织化学。 PCR、染色质免疫沉淀（ChIP）和RNA免疫沉淀（RNA-IP）分析。 目的2：确定这种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、细胞和生物化学方法来确定 lncRNA控制心脏中染色质调节因子的分子功能。的方法 包括RNA-IP、ChIP、定量PCR、电迁移率变动分析、基于游离体报道基因 分析和核小体组装。 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， iPS衍生的心脏细胞以证明lncRNA复合物的存在。同时，我们将使用分子， 细胞和生物化学方法来确定人lncRNA复合物的功能。的方法 包括RNA-IP、少量组织ChIP分析、定量PCR、电迁移率改变 分析、基于附加体的报告基因分析和基于iPS的技术。