microRNA Uncoupling of Protein and Transcript Expression in Liver Regeneration

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控提供了一个独特的系统。这一非凡的过程允许从一系列疾病状态中恢复，包括手术和化学损伤，并依赖于正常静止的细胞重新进入复制。MicroRNAs是新近发现的一种约22nT的非编码RNA，是通过基因表达调控基因的关键效应物。本研究的主要目的是明确microRNAs在70%肝部分切除(PH)后再生大鼠肝脏基因表达调控中的作用。我们的假设是，microRNAs是肝脏再生所必需的，并主要负责在PH后观察到的蛋白质和转录表达的解偶联。第一个具体目标是调查米娜生物发生是否对肝脏再生是必要的。这将通过特定的击倒在MINA加工中的Disher和在RNA诱导的沉默复合体(RISC)中的Ago2的关键成分来完成。利用siRNAs、反义寡核苷酸或基于质粒的表达系统，将在体内进行Dier或Ago2的敲除。MINA水平将通过基因芯片分析在PH后的大鼠体内进行定量。第二个具体目标将描述在肝再生过程中miRNAs与多聚体的关联以及导致其多聚体分布的机制(S)。基于miRNAs跟随mRNAs的假设，我们将比较不同多聚体和非多聚体部分中的整体mRNA和MINA谱。生物信息学将被用来将miRNAs与它们预测的mRNA靶基因联系起来。我们将详细研究c-myc和p53的mRNA活性以及其他已证实的靶向mRNAs的表达和调控。第三个特定目标是确定肝再生过程中直接参与mRNA和蛋白质水平解偶联的miRNAs。我们将通过微阵列分析来描述与肝脏再生相关的miRNAs，并确定miRNAs水平的调节机制。利用生物信息学，我们将选择在肝再生过程中miRNAs解偶联的可能的mRNA靶点。最后，我们将通过2‘-O-甲氧基乙基硫代磷酸反义寡核苷酸敲除选定的miRNAs来证明其功能，并确定其对蛋白质表达的影响。该研究项目的长期目标是建立一个由microRNAs在肝脏再生过程中对mRNA进行转录后调控的模型。建议的研究将为我们理解细胞生长和控制肝脏损伤恢复能力中的基因表达提供重要的新信息。 项目简介：在成年动物中，肝脏是为数不多的、通常处于静止状态的组织之一，它有能力通过物理、感染性或肝毒性损伤来应对细胞损失而再生。最具代表性的肝再生模型是大鼠70%肝部分切除(PH)。在保持完好的肝叶中，大多数肝细胞迅速重新进入生长周期并开始复制。肝脏肿块的恢复主要是由功能因素而不是解剖因素控制的，而且是由残留组织的生长引起的。PH后肝脏的代偿性再生是一个精确的、高度调控的过程，它表现出非常明确的基因表达的时间模式。这一非凡的生理学模型来自于生物控制的精心安排的平衡，这些生物控制在器官的完整大小重建时维持正常的肝功能和结构。这项拨款建议基于三个特定的目标，旨在解决这一假说并阐明microRNAs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角色。所提出的研究将为理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microRNAs在损伤后肝脏再生中所起的作用。最终，这些结果可能为旨在促进肝脏再生和加速从疾病状态中恢复的新疗法提供基础。

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.