Regulation of microRNA Biogenesis in Stem Cells

 描述(申请人提供)：干细胞中微小RNA生物发生的调节。该项目旨在了解胚胎干细胞(ESC)中微RNA(MiRNA)加工的调控机制。胚胎干细胞具有显著的分化为各种细胞类型的能力，对多种退行性疾病具有潜在的治疗价值。然而，胚胎干细胞生物学的分子基础仍然不清楚。MiRNAs作为基因表达的负调控因子，在正常发育过程中对不同的细胞命运决定至关重要；miRNA调控失调与包括癌症在内的几种疾病有关。越来越多的人认识到转录后机制在控制miRNA表达方面发挥着重要作用。Lin28a选择性抑制ESCs中let-7miRNA的生物发生。这一途径有助于维持未分化的细胞状态，并经常在癌症中被重新激活。尽管在阐明Lin28介导的let-7表达调控机制方面已经取得了很大进展，但仍存在一些悬而未决的问题。目的1探讨Lin28/let-7通路的调控。生化和基于细胞的分析相结合，将探索let-7调控的分子决定因素，并将使用最先进的基因工程来探索Lin28/let-7发育开关机制在ESCs中的影响。将使用多种方法来研究Lin28b抑制let-7表达的机制，并探索该途径可能的TUTase依赖性。体外结合和基于细胞的分析将被用来研究已鉴定的lncRNA对Lin28/let-7通路的调节。目的2重点了解控制miR-17~92生物发生的转录后机制。MiR-17~92的精确调控对正常发育至关重要，该簇中某些miRNAs的过度表达是致癌的。新的数据暗示了微处理器上游的一个新的生物发生步骤，它控制着ESCs中miR-17~92的表达。将使用各种生化和细胞分析来测试这一点，并将揭示控制ESCs中miR-17~92表达的RNA特征和调控因素。阐明Lin28/let-7通路和miR-17~92调控的分子细节有望影响生物学和疾病的广泛领域，揭示新的干细胞调节因子，并为治疗癌症、肥胖和糖尿病提供策略。

 DESCRIPTION (provided by applicant): Regulation of microRNA biogenesis in stem cells. This project aims to understand the mechanisms by which microRNA (miRNA) processing is regulated in embryonic stem cells (ESC). ESCs have the remarkable capacity to differentiate into all cell types and are of potential therapeutic value for numerous degenerative diseases. However, the molecular foundations of ESC biology remain poorly defined. Functioning as negative regulators of gene expression, miRNAs are critical for diverse cell fate decisions during normal development; miRNA dysregulation is linked with several diseases including cancer. It is increasingly well appreciated that posttranscriptional mechanisms play an important role controlling miRNA expression. Lin28a selectively represses let-7 miRNA biogenesis in ESCs. This pathway helps maintain an undifferentiated cell state and is often reactivated in cancer. Although much progress has been made elucidating the mechanism of the Lin28-mediated control of let-7 expression, several outstanding questions remain. Aim 1 will explore the regulation of the Lin28/let-7 pathway. A combination of biochemical and cell-based assays will explore the molecular determinants for let-7 regulation and will use state-of-the-art genetic engineering to explore the impact of a Lin28/let-7 developmental switch mechanism in ESCs. A variety of approaches will be used to investigate the mechanism by which Lin28b represses let-7 expression and to explore the possible TUTase-independence of this pathway. In vitro binding and cell-based assays will be employed to investigate the regulation of the Lin28/let-7 pathway by an identified lncRNA. Aim 2 is focused on understanding the posttranscriptional mechanisms controlling miR-17~92 biogenesis. The precise control of miR-17~92 is essential for normal development and overexpression of certain miRNAs from this cluster is oncogenic. New data implicate a novel biogenesis step upstream of Microprocessor that controls miR-17~92 expression in ESCs. A variety of biochemical and cellular assays will be used to test this and will reveal the RNA features and regulatory factors that control miR-17~92 expression in ESCs. Illuminating the molecular details of the Lin28/let-7 pathway and miR-17~92 regulation promises to impact broad areas of biology and disease, reveal novel stem cell regulatory factors, and offer strategies for therapeutic targeting to treat cancer, obesity, and diabetes.