MICRO RNA TARGETING OF NORMAL & LEUKEMIA STEM- PROGENITOR CELLS

项目4：以正常和白血病干细胞为靶点的microRNA(Civin) MicroRNAs与靶mRNAs中的3‘非翻译区结合，下调蛋白质的翻译。 由于microRNAs与其目标mRNAs并不完全碱基配对，因此它们可以阻止蛋白质翻译 许多mRNAs是调节细胞功能的强大开关。将我们的长期研究扩展到 造血和干细胞生物学的调节，我们描述了在造血祖细胞中的microRNA表达 细胞(HSPC)。我们将这一数据与人类HSPC mRNA表达结果相结合， 将microRNA-mRNA靶向预测输入一个新的数据库，该数据库预测某些HSPC表达 MicroRNAs(HE-microRNAs)靶向于几个对造血至关重要的mRNAs。关于这个信息学 在此基础上，我们建立了一个microRNA控制造血的模型，在该模型中，许多基因特定于 早期的HSPC表达造血细胞分化，但HE-microRNAs抑制其分化。为 几个靶向的mRNAs，然后我们实验证明翻译实际上减少了 MicroRNA。MIR-155有效地减少了正常人HSPC的髓系和红系集落形成，并 MIR-16选择性抑制红细胞生成。 由于我们研究的细胞包括稀有干细胞和不同阶段的祖细胞，我们在 目标1，将我们的HSPC microRNA图谱扩展到更高纯度的原代和人类 小鼠HSPC和原代人急性髓系白血病(AMI)细胞。在目标2中，我们将确定是否 正如我们的模型预测的那样，选定的单个microRNA在实验上抑制了初级HSPC的发育， 如果这些微小RNA影响急性心肌梗死干细胞和白血病发生。我们将确定它的蛋白质 合成被每个具有功能活性的microRNA抑制，从而抑制了 有造血作用。在我们研究的这一点上，至少出现了两个这样的microRNAs，mir-16和mir-155 成为正常造血干细胞和癌症干细胞的新调节者。 相关性：我们认为，阻碍许多癌症患者治愈的一个主要障碍是我们无法治愈他们 今天的治疗方法是我们未能有效地攻击和消除癌症干细胞。具体地说，我们认为 白血病更有效的治疗方法的开发取决于对REARE调节的更充分的理解 正常和白血病的造血干细胞。该项目中的研究调查了新的 被称为microRNAs的调节分子，似乎在之前的控制中扮演着意想不到的、强有力的角色 血液形成和白血病的可能性。了解这些造血调节microRNA的作用 可能为正常干细胞的扩增和白血病的治疗提供新的靶点。

Project 4: MicroRNA targeting of normal and leukemia stem-progenitor cells (Civin) MicroRNAs bind to 3' untranslated region sites in target mRNAs to down-regulate translation to protein. Since microRNAs do not base-pair exactly with their target mRNAs, they can block protein translation of many mRNAs and serve as powerful switches to regulate cell functions. To extend our long-term studies on regulation of hematopoiesis and stem cell biology, we profiled microRNA expression in hematopoietic stemprogenitor cells (HSPCs). We combined this data with human HSPC mRNA expression results and microRNA-mRNA target predictions into a novel database which predicted that certain of the HSPCexpressed microRNAs (HE-microRNAs) targeted several mRNAs critical to hematopoiesis. On this informatic basis, we formulated a model for microRNA control of hematopoiesis in which many genes specifying hematopoietic differentiation are expressed by early HSPCs, but held in check by HE-microRNAs. For several target mRNAs, we then demonstrated experimentally that translation is actually decreased by microRNAs. Mir-155 potently reduced myeloid and erythroid colony formation of normal human HSPCs, and mir-16 selectively inhibited erythropoiesis. Since the cells that we studied include rare stem cells and various stages of progenitors, we propose in Aim 1, to expand our microRNA profiles of HSPCs to more highly purified subsets of primary human and mouse HSPCs and primary human acute myeloid leukemia (AMI) cells. In Aim 2, we will determine if selected individual microRNAs experimentally inhibit development of primary HSPCs, as our model predicts, and if these microRNAs affect AMI stem cells and leukemogenesis. We will determine the proteins whose synthesis is inhibited by each functionally-active microRNA and thereby the molecular mechanisms of the hematopoietic effects. At this point in our studies, at least 2 of these microRNAs, mir-16 and mir-155 appear to be new regulators of normal hematopoietic and cancer stem cells. Relevance: We suggest that a major barrier impeding cure of the many cancer patients whom we cannot cure today is our failure to effectively attack and eliminate cancer stem cells. Specifically, we believe that development of more effective therapies in leukemia hinges on fuller understanding of the regulation of rare normal and leukemic hematopoietic stem cells. The studies in this project investigate the effects of new regulatory molecules called microRNAs, which appear to play previously unexpected, potent roles in control of blood formation and leukemia. Understanding the actions of these hematopoietic-regulatory microRNAs may provide new targets, for both expansion of normal stem cells and leukemia therapy.