Mechanisms of miRNA regulation of early embryonic development

在胚胎的发育过程中，细胞要经历一个阶段，在这个阶段，细胞有可能产生形成新生有机体的所有细胞类型和组织。这种潜能被称为多能性，这些多能性的细胞是干细胞，在再生医学上有很大的希望。这一前景不仅是因为它们未来在细胞治疗中的临床应用，还因为现在可以产生针对患者的多能细胞，用于“培养皿中的疾病”方法，以了解人类疾病。干细胞疗法正被用于治疗从糖尿病到神经退行性疾病的各种疾病，而基于干细胞的机制被认为是不同形式癌症的基础。处于多能状态的细胞需要对它们将产生的细胞类型做出选择，并通过严格的质量控制，因为只有那些没有缺陷的细胞才能存活。这意味着多能性细胞受到严格的调控，理解这种调控不仅对了解胚胎如何形成很重要，而且对利用多能性细胞进行再生医学也很重要。本项目旨在了解多能细胞中细胞存活和分化程序的调控。microrna是一种抑制基因表达的小非编码rna，在过去十年中已成为许多过程的关键调节因子，包括发育、细胞增殖、细胞凋亡和许多疾病，包括癌症。在提出这一建议的初步工作中，我们发现microrna是多能细胞存活和分化为神经细胞类型所必需的。因此，这些观察结果直接暗示了microrna对多能细胞的调控。了解microRNAs如何调节细胞存活和多能状态的分化是本项目的主要重点。作为第一步，我们将利用小鼠遗传学和多能干细胞系的“培养皿”方法来删除Dicer，这是对microRNA生成至关重要的蛋白质。通过研究Dicer缺失和microRNA缺失对胚胎和多能干细胞系的影响，我们将能够准确地揭示microRNA在多能状态调节中的作用。根据它们抑制的基因，microrna被分成不同的家族，一个家族中的microrna被认为是针对相同的基因。我们在该项目的初步工作中发现miR-291a-3p、miR-17、miR-19和miR-92家族的microrna是参与多能细胞存活和分化调控的强有力候选者。由于这些原因，我们的工作将集中在这些microrna上，并测试哪些家族可以挽救多能干细胞中由于microrna耗竭而引起的缺陷。最后，我们的目标是确定调控多能细胞存活和分化的microrna靶向哪些基因。这将建立调控多能性的microRNA-target相互作用。鉴于microRNA抑制基因表达，我们将研究当细胞被给定的microRNA处理时，哪些基因的表达会减少。鉴于许多细胞类型都充满了microRNA，我们将使用Dicer缺陷多能干细胞进行这些研究，因为它们代表了一种无microRNA的细胞类型。预测microRNA靶标的生物信息学程序将用于从实验鉴定的microRNA靶标基因中生成可能的microRNA靶标基因列表。最佳候选基因将通过microRNA靶位点的突变来验证。这将确定哪些交互在功能上是重要的。

During the development of an embryo, cells transit through a stage where they possess the potential to give rise to all the cell types and tissues that will form the newborn organism. This potential is called pluripotency, and those cells that are pluripotent are stem cells and hold great promise for regenerative medicine. This promise is not only due to their future clinical applications in cellular therapies, but also because now patient specific pluripotent cells can be generated for "disease-in-a-dish" approaches to understand human diseases. Stem cell therapies are being developed for diseases ranging from diabetes to neurodegenerative disorders and a stem cell based mechanism is thought to underlie different forms of cancer. The cells that are in the pluripotent state need to make choices regarding the cell type that they will give rise to and pass strict quality controls, as only those that are not defective will survive. This implies that pluripotent cells are under tight regulation and understanding this regulation is important not only to gain insight into how an embryo forms but as well to be able to use pluripotent cells for regenerative medicine. This project aims to understand the regulation of the cell survival and differentiation programs in pluripotent cells.MicroRNAs are small non-coding RNAs that inhibit gene expression and have emerged over the past decade as key regulators involved in many processes including development, cell proliferation, apoptosis and in numerous diseases, including cancer. In the preliminary work leading up to this proposal we have found that microRNAs are required for the survival of pluripotent cells and for these cells to differentiate into neural cell types. These observations therefore have directly implicated microRNAs in the regulation of pluripotent cells. Understanding how microRNAs regulate cell survival and differentiation of the pluripotent state is the main focus of this project.As a first step we will use mouse genetics as well as pluripotent stem cell lines for "in a dish" approaches to delete Dicer, that is the protein that is essential for microRNA generation. By studying the effects of Dicer deletion, and therefore microRNA depletion, in the embryo and in pluripotent stem cell lines we will be able to uncover precisely what the roles of microRNAs are in the regulation of the pluripotent state.MicroRNAs fall into families according to the genes that they inhibit, those within a family are thought to target the same genes. Our preliminary work to this project identified microRNAs of the miR-291a-3p, miR-17, miR-19 and miR-92 families as strong candidates for being involved in the regulation of the survival and differentiation of pluripotent cells. For these reasons our work will focus on these microRNAs and test which families can rescue the defects caused by the depletion of microRNAs in pluripotent stem cells.Finally, we aim to identify what genes are targeted by the microRNAs that regulate pluripotent cell survival and differentiation. This will establish the microRNA-target interactions that regulate pluripotency. Given that microRNAs inhibit gene expression, we will study what genes decrease in expression when cells are treated with a given microRNA. Given that many cell types are saturated with microRNAs, we will use Dicer deficient pluripotent stem cells for these studies, as they represent a microRNA free cell type. Bioinformatic programs to predict microRNA targets will be used to generate a list of likely microRNA target genes from those identified experimentally. The best candidate genes will be validated by mutation of the microRNA target site in it. This will establish which interactions are functionally important.