Genetic Analysis of MicroRNA Functions in Skin Stem Cells in Vivo

描述(申请人提供)：哺乳动物的皮肤及其附属物作为身体最外层的屏障，保护内部器官免受环境危害，并将必要的液体保留在体内。哺乳动物皮肤的动态平衡和完整性是由居住在不同皮肤隔间的多个祖细胞和干细胞群体维持的。特别是，位于隆起区的多能毛囊干细胞(HfSCs)在正常的毛发周期和表皮创面愈合过程中维持毛囊(Hf)。了解调控hfSCs自我更新和激活的分子机制是将这些迷人的细胞用于再生医学的基本前提。在这些机制中，microRNAs(MiRNAs)是一类小的、非编码的RNAs，在不同细胞类型和组织中对哺乳动物的基因调控起着至关重要的作用。与有效激活或抑制基因表达的转录机制不同，miRNAs在转录后水平调节mRNAs的蛋白质生产，通常功能是微调转录组的输出。尽管对单个靶标进行了适度的调控，但miRNAs广泛地调控着大量(60%)的基因，并在广泛的生物过程中发挥着重要作用。在哺乳动物皮肤中，整个miRNA通路在胚胎皮肤发育和成人HF谱系维持中的关键功能已经被很好地认识到。与此形成鲜明对比的是，目前还没有关于成人皮肤中hfSCs自我更新所需的任何单个miRNA的报道。因此，miRNA介导的hfSCs调控的潜在机制在很大程度上仍不清楚。为了解决这些重要问题，我们建议将重点放在miR-205上，它是hfSCs中表达最高和功能最重要的miRNA，并研究其在成年hfSCs自我更新和迁移中的作用(目标1和2)。为了提供这些功能的机械性见解，我们将把miRISC及其相关mRNA靶点的生化纯化应用于WT和miR-205基因敲除的hfSCs，并以HFSC特异性的方式识别所有与miR-205相关的靶点。该项目的长期目标是阐明单个miRNAs的功能，并为这些分子在动态平衡和应激条件下的机制提供见解，包括hfSCs的损伤反应和肿瘤发生。综上所述，这里提出的研究，如果成功，将显著增强我们对单个miRNAs在成年hfSCs中的功能的了解。在正常和压力条件下从这些研究中获得的知识将为操纵miRNAs和将这些干细胞用于再生医学铺平道路。

DESCRIPTION (provided by applicant): Mammalian skin and its appendages function as the outermost barrier of the body to protect inner organs from environmental hazards and keep essential fluids within the body. Homeostasis and integrity of mammalian skin are maintained by multiple progenitor and stem cell populations residing in distinct skin compartments. In particular, multipotent hair follicle stem cells (HFSCs) located in the bulge region maintain hair follicles (HFs) during normal hair cycle and epidermal wound healing. Understanding molecular mechanisms that regulate self-renewal and activation of HFSCs is a fundamental prerequisite to exploit these fascinating cells for regenerative medicine. Among the mechanisms, microRNAs (miRNAs) are a class of small, noncoding RNAs that take essential roles in mammalian gene regulation in diverse cell types and tissues. Unlike transcriptional mechanisms that potently activate or suppress gene expression, miRNAs regulate protein production of mRNAs at post-transcriptional levels and usually function to fine tune the output of the transcriptome. Despite modest regulation of individual targets, miRNAs broadly modulate a large number (60%) of genes and play important roles in a wide range of biological processes. In mammalian skin, the critical functions of the entire miRNA pathway in both embryonic skin development and maintenance of adult HF lineages have been well appreciated. In sharp contrast, there is no report identifying any individual miRNA that is required for the self-renewal of the HFSCs in adult skin. Consequently, the underlying mechanism of miRNA-mediated regulation in the HFSCs remains largely unknown. To address these important issues, we propose to focus on miR-205, the most highly expressed and functionally important miRNA in the HFSCs, and examine its roles in self- renewal and migration of adult HFSCs (Aim 1 and 2). To provide mechanistic insights for these functions, we will apply biochemical purification of miRISC and its associated mRNA targets to the WT and miR-205 knockout HFSCs and identify all miR-205-associated targets in an HFSC- specific manner. The long-term goal of this project is to elucidate individual miRNAs' functions and provide mechanistic insights to these molecules during homeostasis and stressed conditions including injury response and tumorigenesis in the HFSCs. Taken together, studies proposed here, if successful, will significantly enhance our knowledge about individual miRNAs' functions in adult HFSCs. The knowledge gained from these studies under normal and stressed conditions will pave the way to manipulate miRNAs and utilize these stem cells for regenerative medicine.