Functional interaction between the mir11~998 intronic microRNA cluster and Retinoblastoma tumor suppressor pathway

描述(由申请人提供)：视网膜母细胞瘤(RB)肿瘤抑制通路的失活是人类癌症的早期强制性事件。Rb途径的关键靶点之一是调控细胞增殖和凋亡的E2F转录因子家族。 E2F和PRB家族哺乳动物成员的重叠和冗余功能经常使其体内功能的鉴定复杂化。由于Rb途径的高度保守性和果蝇对遗传分析的适应性，果蝇等模式生物为哺乳动物系统的补充研究提供了有吸引力的替代方案。有趣的是，果蝇E2F基因dE2f1含有一个内含子microRNA簇mir11~998。这种排列为miRNA及其宿主基因之间可能的功能相互作用奠定了基础。令人惊讶的是，尽管超过一半的脊椎动物miRNAs是内含子的，这个问题在很大程度上仍然没有被认识到，因此这类miRNAs很少在它们所在的基因的背景下进行研究。我们最近发现mir11~998簇与E2F依赖性细胞凋亡的调控密切相关。MIR-11直接调节凋亡的E2F靶标的表达，以限制辐射诱导的细胞凋亡。与miR-11不同，miR-998通过不同的机制运行。在初步实验中，我们发现miR-998通过调节存活的EGFR信号来调节E2F依赖的细胞凋亡。我们会以这些初步研究结果为基础，建立 嵌入dE2f1基因的mir11~998簇调控的功能意义在目标1中，我们将验证dCbl作为miR-998的直接靶点来介导其在果蝇EGFR信号转导中的作用。在目标2中，我们将把我们的工作扩展到哺乳动物细胞，并将评估哺乳动物miR-998同源物miR-29和EGFR信号之间的联系。在AIM3中，我们将研究mir11~998簇及其宿主基因dE2f1的共同调控。我们将使用新的敲入等位基因来确定miR-11和miR-998之间的相互依赖在簇内的功能意义。总体而言，这项研究将解决嵌入的miRNAs和它们的宿主基因之间的很大程度上未被认识的关系，并将评估这种相互作用在Rb肿瘤抑制途径背景下的意义。值得注意的是，miRNAs已知可以影响各种生物学过程，并在癌症等人类恶性肿瘤中发挥关键作用。因此，该项目成功完成后产生的知识可能会刺激将miRNA调节与E2F联系起来的进一步研究，并可能有助于确定新的治疗靶点。

DESCRIPTION (provided by applicant): Inactivation of Retinoblastoma (Rb) tumor suppressor pathway is an early obligatory event in human cancer. One of the key targets of Rb pathway is the family of E2F transcription factors that regulates cell proliferation and apoptosis. Overlapping and redundant functions of mammalian members of E2F and pRB families often complicate identification of their in vivo function. Model organisms such as Drosophila provide attractive alternatives to complement studies in mammalian systems due to high conservation of Rb pathway and amenability of flies to genetic analysis. Interestingly, Drosophila E2F gene dE2f1 contains an intronic microRNA cluster mir11~998. Such arrangement sets a stage for possible functional interaction between the miRNA and its host gene. Surprisingly, even though more than half of vertebrate miRNAs are intronic this issue remains largely unappreciated and therefore this class of miRNAs is rarely investigated in the context of the gene they reside in. We have recently discovered that the mir11~998 cluster is intimately involved in modulation of E2F dependent apoptosis. miR-11 directly regulates expression of apoptotic E2F targets to limit irradiation induced apoptosis. Unlike miR-11, miR-998 operates through a different mechanism. In preliminary experiments, we have found that miR-998 regulates E2F dependent apoptosis by modulating the prosurvival EGFR signaling. We will build on these initial findings to establish the functional significance of regulation of the mir11~998 cluster embedded into the dE2f1 gene. In Aim 1, we will validate dCbl as a direct target of miR-998 to mediate its effect on EGFR signaling in Drosophila. In Aim 2, we will extend our work to mammalian cells and will evaluate the link between miR-29, a mammalian miR-998 homolog, and EGFR signaling. In Aim3, we will investigate the co-regulation of the mir11~998 cluster and its host gene dE2f1. We will determine the functional significance of interdependence between miR-11 and miR-998 within the cluster using novel knock-in alleles. Overall, this study will address largely unappreciated relationship between embedded miRNAs and their host gene and will evaluate the significance of such interaction in the context of Rb tumor suppressor pathway. Notably, miRNAs are known to influence various biological processes and play a crucial role in human malignancies such as cancer. Therefore the knowledge generated upon successful completion of this project will likely stimulate further studies linking miRNA regulation to E2F and may help in identifying novel therapeutic targets.