microRNA Uncoupling of Protein and Transcript Expression in Liver Regeneration

肝脏再生中蛋白质和转录物表达的 microRNA 解偶联

• 批准号: 7924203
• 负责人: CLIFFORD John STEER
• 金额: $ 48.16万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924203
• 关键词: Address; Affect; Antisense Oligonucleotides; Applications Grants; Architecture; Biogenesis; Bioinformatics; Biological; Biological Assay; Cell Culture Techniques; Cell Proliferation Regulation; Cells; Characteristics; Chemical Injury; Complex; Disease; Equilibrium; Exhibits; Functional RNA; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; Growth; Growth and Development function; Hepatic Mass; Hepatocyte; Injury; Liver; Liver Regeneration; Lobe; Messenger RNA; MicroRNAs; Microarray Analysis; Modeling; Molecular; Natural regeneration; Normal Cell; Oligonucleotides; Operative Surgical Procedures; Organ Size; Partial Hepatectomy; Pattern; Plasmids; Play; Polyribosomes; Population; Process; Proteins; RNA Interference; RNA-Induced Silencing Complex; Rat-1; Rattus; Recovery; Regulation; Research; Research Project Grants; Role; Small Interfering RNA; Small RNA; System; TP53 gene; Techniques; Testing; Tissues; Transcript; Translations; base; c-myc Genes; cell growth; design; expression vector; human DICER1 protein; in vivo; liver function; mRNA Expression; mature animal; novel; phosphorothioate; physiologic model; protein expression; reconstitution; response; restoration; small hairpin RNA; therapy design

DESCRIPTION (provided by applicant): The ability of the liver to regenerate provides a unique system to study the in vivo regulation of cell proliferation and gene expression. This remarkable process allows recovery from a number of disease states, including surgical and chemical injuries, and is dependent on the reentry of normally quiescent cells into replication. MicroRNAs are recently discovered ~ 22 nt non-coding RNAs that are known to be critical effectors of gene regulation via mRNA expression. The main objective of this research project is to define the role of microRNAs in the regulation of gene expression in regenerating rat liver after 70% partial hepatectomy (PH). Our hypothesis is that microRNAs are required for liver regeneration and primarily responsible for the observed uncoupling of protein and transcript expression after PH. The first specific aim is designed to investigate whether mina biogenesis is essential for liver regeneration. This will be done by specific knockdown of critical components of Dicer in mina processing and Ago2 in the RNA Induced Silencing Complex (RISC). Knockdown of Dicer or Ago2 will be carried out in vivo using siRNAs, antisense oligonucleotides or plasmid-based expression-systems. mina levels will be quantitated via microarray analysis in rats after PH. The second specific aim will characterize the association of miRNAs with polysomes during liver regeneration and the mechanism(s) responsible for their polysome distribution. Based on the hypothesis that miRNAs follow mRNAs, we will compare global mRNA and mina profiles in the different polysome and non-polysome fractions. Bioinformatics will be used to correlate miRNAs with their predicted mRNA targets. We will examine in detail the expression and modulation of c-myc and p53 mRNA activity and other verified target mRNAs. The third specific aim is designed to identify miRNAs during liver regeneration that are directly involved in the uncoupling of mRNA and protein levels. We will profile miRNAs associated with liver regeneration via microarray analysis and determine the mechanism by which miRNAs levels are modulated. Using bioinformatics, we will select putative mRNA targets of uncoupling by miRNAs during liver regeneration. Finally, we will establish proof of function for selected miRNAs via their knockdown by 2'-O-methyoxyethyl phosphorothioate antisense `antagomir' oligonucleotides, and determine the effect on protein expression. The long-term goal of the research project is to formulate a model of posttranscriptional regulation of mRNA by microRNAs in liver regeneration. The proposed studies will provide important and new information in our understanding of cell growth and the control of gene expression in the liver's ability to recover from injury. PROJECT NARRATIVE: The liver constitutes one of the few, normally quiescent tissues in the adult animal that has the capacity to regenerate in response to cell loss through physical, infectious or hepatotoxic injury. The best-characterized model of liver regeneration is the 70% partial hepatectomy (PH) in rat. In the lobes that remain intact, the majority of hepatic cells rapidly reenter the growth cycle and begin to replicate. Restoration of liver mass is governed primarily by functional rather than anatomical factors, and occurs by growth of the remnant tissue. The compensatory regrowth of the liver after PH is a precise, highly regulated process which exhibits very defined temporal patterns of gene expression. This remarkable physiologic model results from an orchestrated balance of biological controls, which maintain normal liver function and architecture as the full-size of the organ is reconstituted. This grant proposal is based on three specific aims intended to address the hypothesis and elucidate the role of microRNAs in the regulation of gene expression in regenerating liver. These remarkable small RNA molecules have only recently been discovered; and it is now apparent that they are key players in development and growth of tissues. The proposed studies will provide important information for understanding the control of normal cell growth in vivo. Our goal is to understand the role that microRNAs play in the regeneration of liver after injury. Ultimately, the results may provide the basis for novel therapies designed to promote liver regeneration and accelerate recovery from disease states.

描述（由申请人提供）：肝脏再生的能力为研究细胞增殖和基因表达的体内调节提供了独特的系统。这个非凡的过程允许从许多疾病状态中恢复，包括手术和化学损伤，并且依赖于正常静止细胞重新进入复制。MicroRNA是最近发现的~ 22 nt的非编码RNA，已知其是通过mRNA表达进行基因调控的关键效应子。本研究项目的主要目的是确定microRNA在70%部分肝切除术（PH）后再生大鼠肝脏中基因表达调控中的作用。我们的假设是，微小RNA是肝再生所需的，主要负责观察到的解偶联后PH的蛋白质和转录表达。第一个具体的目的是调查是否米娜生物合成是必不可少的肝再生。这将通过特异性敲除mina加工中的Dicer和RNA诱导沉默复合物（RISC）中的Ago 2的关键组分来完成。Dicer或Ago 2的敲低将使用siRNA、反义寡核苷酸或基于质粒的表达系统在体内进行。将通过微阵列分析定量PH后大鼠中的mina水平。第二个具体目标将表征肝再生期间miRNA与多核糖体的关联以及负责其多核糖体分布的机制。基于miRNA跟随mRNA的假设，我们将比较不同多核糖体和非多核糖体组分中的总体mRNA和mina谱。生物信息学将用于关联miRNA与其预测的mRNA靶标。我们将详细研究c-myc和p53 mRNA活性以及其他经验证的靶mRNA的表达和调节。第三个具体目标是鉴定肝再生过程中直接参与mRNA和蛋白质水平解偶联的miRNA。我们将通过微阵列分析与肝再生相关的miRNAs，并确定miRNAs水平调节的机制。利用生物信息学，我们将选择肝再生过程中miRNA解偶联的假定mRNA靶点。最后，我们将通过2 '-O-甲氧基乙基硫代磷酸酯反义寡核苷酸敲低所选miRNA来建立功能证据，并确定对蛋白质表达的影响。该研究项目的长期目标是制定一个模型，通过microRNA在肝再生中对mRNA进行转录后调控。这些研究将为我们理解细胞生长和肝脏从损伤中恢复的基因表达控制提供重要和新的信息。 项目叙述：肝脏是成年动物中为数不多的正常静止的组织之一，它具有再生能力，以响应由于物理、感染或肝毒性损伤而导致的细胞损失。最具特征的肝再生模型是大鼠70%部分肝切除（PH）。在保持完整的肝叶中，大多数肝细胞迅速重新进入生长周期并开始复制。肝脏质量的恢复主要由功能因素而不是解剖因素决定，并且通过残余组织的生长而发生。PH后肝脏的代偿性再生是一个精确的、高度调节的过程，其表现出非常明确的基因表达时间模式。这一非凡的生理模型源于生物控制的协调平衡，在器官的全尺寸重建时维持正常的肝功能和结构。这项拨款提案基于三个具体目标，旨在解决这一假设，并阐明microRNA在再生肝脏基因表达调控中的作用。这些引人注目的小RNA分子最近才被发现;现在很明显，它们是组织发育和生长的关键参与者。这些研究将为了解体内正常细胞生长的控制提供重要信息。我们的目标是了解microRNA在损伤后肝脏再生中的作用。最终，这些结果可能为旨在促进肝脏再生和加速疾病状态恢复的新疗法提供基础。

项目成果

Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture.

• DOI: 10.1002/hep.24712
• 发表时间: 2012-02
• 期刊: HEPATOLOGY
• 影响因子: 13.5
• 作者: Chen, Yixin;Wong, Philip P.;Sjeklocha, Lucas;Steer, Clifford J.;Sahin, M. Behnan
• 通讯作者: Sahin, M. Behnan