Investigating microRNA Function in Homeostasis, Regeneration and Cancer

摘要 基因表达的精确时间和空间控制对于确保正常发育和 体内平衡，并通过多种调控机制，在每一步从基因 转录到蛋白质降解。microRNAs（miRNAs）是一类高度保守的 在转录后水平调节基因表达的调节性短非编码RNA。 从机制上讲，miRNAs抑制它们的靶点，作为一个大的多组分核糖核蛋白的一部分， miRNA诱导的沉默复合物（miRISC），包括Argonaute蛋白（AGO）和 TNRC6蛋白家族。 miRISC的组装和活性在体内是如何调节的，目前还知之甚少，直到最近， 默认miRNAs在所有细胞类型中都具有组成性活性。几个小组最近的工作，包括 我们的研究挑战了这一假设，揭示了miRISC的组装在体内受到严格的调控， 这表明在许多成年组织和静止细胞中，大量的miRNA结合的AGO可能是由细胞内的细胞分泌的。 蛋白质不与靶mRNA结合，并且不参与它们的抑制。 为什么许多成年组织和静止细胞含有高水平的功能失活的miRNA， 除了促有丝分裂的线索，是否还有其他干扰因素，包括癌症、衰老、感染和 炎症调节miRISC组装和活性是该领域中两个主要的未回答的问题 也是这次拨款申请的重点 作为解决这两个问题的第一步，我们提出了一系列利用小说的实验， 我们最近产生的基因工程小鼠品系允许在体内控制miRISC组装 以时间和空间受控的方式。我们将使用这种新的小鼠品系来确定 在正常组织稳态、发育和组织再生过程中的miRISC活性（目的1）。 在目标2中，我们将使用它来直接检验miRISC活性是肿瘤进展所需的假设， 我们将确定miRISC抑制作为一种新的抗癌策略的潜力。 最后，在目标3中，我们将利用我们开发的一种新的小鼠品系来获得机制见解 miRNAs是如何控制这些基本过程的。 该项目的成功完成将极大地推进我们对miRNA介导的基因在细胞内的作用的理解， 抑制哺乳动物，并可能导致新的抗癌策略的发展。

ABSTRACT Precise temporal and spatial control of gene expression is essential to ensure normal development and homeostasis and is achieved through multiple regulatory mechanisms acting at every step from gene transcription to protein degradation. MicroRNAs (miRNAs), constitute a large class of highly conserved regulatory short non-coding RNAs that modulate gene expression at the post-transcriptional level. Mechanistically, miRNAs repress their targets as part of a large multicomponent ribonucleoprotein known as the miRNA-Induced Silencing Complex (miRISC), which include Argonaute proteins (AGO) and members of the TNRC6 family of proteins. How miRISC assembly and activity are regulated in vivo remains poorly understood and until recently it was tacitly assumed that miRNAs are constitutively active in all cell types. Recent work from several groups, including ours, has challenged this assumption, revealing that assembly of the miRISC is tightly regulated in vivo, and suggesting the possibility that in many adult tissues and in quiescent cells the bulk of miRNA-bound AGO proteins are not bound to target mRNAs, and are not engaged in their repression. Why many adult tissues and quiescent cells contain high levels of functionally inactive miRNAs and whether, in addition to mitogenic cues, other perturbations—including cancer, aging, infection, and inflammation—regulate miRISC assembly and activity are two major unanswered questions in the field and the main focus of this grant proposal. As a first step to address these two questions we propose a series of experiment that take advantage of a novel genetically engineered mouse strain we have recently generated that allows to control miRISC assembly in vivo in a temporally and spatially controlled fashion. We will use this novel mouse strain to determine the requirement for miRISC activity during normal tissue homeostasis, in development, and during tissue regeneration (Aim 1). In Aim 2, we will use it to directly test the hypothesis that miRISC activity is required for tumor progression and tumor maintenance in vivo and we will determine the potential of miRISC inhibition as a novel anticancer strategy. Finally, in Aim 3 we will take advantage of a novel mouse strain we have developed to gain mechanistic insights into how miRNAs control these essential processes. Successful completion of this project will greatly advance our understanding of the role of miRNA-mediated gene repression in mammals and could lead to the development of novel anti-cancer strategies.