Investigating microRNA Function in Homeostasis, Regeneration and Cancer

摘要 精确的基因表达的时间和空间控制对于确保正常发育和 动态平衡是通过作用于基因的每一步的多种调节机制来实现的 转录到蛋白质降解。MicroRNAs(MiRNAs)，构成一大类高度保守的 在转录后水平调节基因表达的调节性非编码短RNA。 从机制上讲，miRNAs作为一种大型多组分核糖核蛋白的一部分抑制它们的靶标 MiRNA诱导沉默复合体(MiRISC)，包括ArgAerte蛋白(AGO)和 TNRC6蛋白家族。 MiRISC在体内的组装和活性是如何被调控的仍然知之甚少，直到最近才有所了解 心照不宣地假设miRNAs在所有类型的细胞中都是结构性活跃的。几个小组最近的工作，包括 我们的，挑战了这一假设，揭示了miRISC的组装在体内受到严格调控，并且 这表明在许多成人组织和静止细胞中，大量的miRNA结合的AGO 蛋白质不与mRNAs结合，也不参与它们的抑制。 为什么许多成人组织和静止细胞含有大量功能不活跃的miRNAs和 除了有丝分裂信号外，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两大悬而未决的问题 以及这项拨款提案的主要重点。 作为解决这两个问题的第一步，我们提出了一系列实验，这些实验利用了一本小说 我们最近培育的基因工程小鼠品系可以在体内控制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需求 在正常组织动态期、发育期和组织再生期间的miRISC活性(目标1)。 在目标2中，我们将使用它来直接测试miRISC活性是肿瘤进展所必需的假设，并且 肿瘤的体内维持，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械洞察力 MiRNAs如何控制这些重要的过程。 该项目的成功完成将极大地促进我们对miRNA介导的基因作用的理解。 在哺乳动物中的抑制，并可能导致新的抗癌策略的开发。

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.