RNA binding proteins and intestinal stem cells

描述（由申请人提供）：肠上皮是体内任何组织中周转率最高的组织之一。因此，肠上皮自我更新是一个重要的生理过程，在很大程度上依赖于干细胞。由干细胞产生的祖细胞或中转扩增细胞最终重建肠上皮的分化细胞。由于这些干细胞在自我更新中的重要性，必须保护它们免受基因毒性损伤，如辐射。RNA结合基序3 （RNA binding motif 3, RBM3）是一种在肠上皮细胞中表达的RNA结合蛋白。实验室先前的工作表明，RBM3通过RNA稳定性和翻译上调Cyclin D1、VEGF和COX-2等基因。此外，RBM3对细胞增殖、化学抗性、辐射抗性、锚定独立生长、抗有丝分裂突变和血管生成均有显著影响。Notch1是一种跨膜信号受体。激活后，Notch1被裂解释放胞内结构域，该结构域易位到细胞核并诱导Notch1调控基因的转录。多项研究表明，Notch信号通路在调节干细胞和祖细胞结构中起着关键作用。我们的初步数据表明，RBM3可以增加表达假定的干细胞标记物DCLK1的细胞数量。我的初步研究表明，这些细胞具有显著的增殖潜力，并显示出极大的抗辐射能力。在这个提议中，我们假设RBM3通过改变Notch信号通路增加了干细胞样细胞的百分比，并且这些RBM3诱导的干细胞对辐射具有显著的抗性。在目标1中，我们将验证DCLK1和其他假定的干细胞标记物是RBM3诱导因子，与细胞群体中增加的“干性”相关。我们将使用诱导型RBM3过表达系统和测定肠上皮细胞系中干细胞标记物表达的变化。我们还将确定这些细胞显示干细胞样表型。在目标2中，我们将阐明RBM3在影响Notch信号级联中的作用，并描述Notch信号通路与目标干细胞标记物之间的相互作用。我们计划通过使用Notch信号通路的小分子抑制剂以及sirna介导的Notch通路蛋白的敲低来做到这一点。最后，在目标3中，我们将确定RBM3对体内干细胞表型的影响。这将通过使用RBM3过表达细胞和RBM3诱导干细胞生成肿瘤异种移植物来实现。将检测异种移植物的增殖、产生异质组织的能力和抗辐射能力。这些研究将揭示RBM3在异质细胞群中增加干细胞样表型的机制，以及这些RBM3诱导的干细胞表型如何产生对辐射的抗性。我们相信这些研究将是了解RBM3在调节肠道干细胞生理中的作用的第一步。

DESCRIPTION (provided by applicant): The intestinal epithelium has one of the highest turnovers of any tissue in the body. As such, intestinal epithelial self renewal is an important physiologic process which relies heavily on the stem cell. Progenitor or transit amplifying cells, arising from the stem cell ultimately reconstitute the differentiated cells of the intestinal epithelium. Due to the importance of these stem cells in self renewal, they must be protected from genotoxic insults such as radiation. RNA binding motif 3 (RBM3) is an RNA binding protein that is expressed in the intestinal epithelial cells. Prior work from the laboratory has shown that RBM3 upregulates several genes such as Cyclin D1, VEGF and COX-2 through RNA stability and translation. Furthermore, RBM3 had significant effects on cell proliferation, chemoresistance, radiation resistance, anchorage independent growth, resistance to mitotic catastrophe, and angiogenesis. Notch1 is a transmembrane signaling receptor. Upon activation, Notch1 is cleaved releasing the intracellular domain, which translocates to the nucleus and induces transcription of Notch-regulated genes. Several studies show that the Notch signaling pathway is a key player in regulating stem cell and progenitor cell hierarchy. Our preliminary data indicate that RBM3 can increase the number of cells expressing the putative stem cell marker DCLK1. My preliminary studies suggest that these cells have significant proliferative potential and show a tremendous increase in radiation resistance. In this proposal, we hypothesize that RBM3 increases the percentage of stem-like cells through altering the Notch signaling pathway and that these RBM3-induced stem-like cells have significant resistance to radiation. In aim 1, we will validate DCLK1 and other putative stem cell markers as RBM3 induced factors that correlate with increased "stemness" within a cell population. We will use an inducible RBM3 overexpressing system and assay for changes in the expression of stem cell markers within intestinal epithelial cell lines. We will also determine that these cells display a stem like phenotype. In aim 2, we will elucidate the role of RBM3 in affecting the Notch signaling cascade and characterize the interplay between the Notch signaling pathway and target stem cell markers. We plan to do this by using small molecule inhibitors of the Notch signaling pathway as well as siRNA-mediated knockdown of Notch pathway proteins. Finally in aim 3, we will determine the effect that RBM3 has on the stem cell phenotype in vivo. This will be performed by generating tumor xenografts using RBM3 overexpressing cells and RBM3 induced stem cells. The xenografts will be assayed for proliferation, ability to generate heterogenous tissue, and radiation resistance. These studies should reveal a mechanism by which RBM3 increases a stem-like phenotype within a heterogenous cell population and how these RBM3 induced stem cell phenotype generates resistance to radiation. We believe that these studies will be the first step in understanding the role for RBM3 in regulating intestinal stem cell physiology. PUBLIC HEALTH RELEVANCE: The intestinal stem cell is a key point in the self renewal pathway. Because the intestine is an organ most affected by exposure to radiation it is important to understand the stem cell physiology responsible for regenerating the intestine following an insult. The current study will further our understanding of how cells acquire and maintain a stem like phenotype and how this stem like phenotype can confer resistance to genotoxic insults such as radiation.