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（Retinoblastoma，Rb）肿瘤抑制通路的失活是人类肿瘤发生的早期必然事件。Rb通路的关键靶点之一是调节细胞增殖和凋亡的E2 F转录因子家族。 E2 F和pRB家族的哺乳动物成员的重叠和冗余功能通常使其体内功能的鉴定复杂化。模式生物，如果蝇，提供了有吸引力的替代，以补充研究在哺乳动物系统中，由于高度保守的Rb途径和顺从的苍蝇遗传分析。有趣的是，果蝇E2 F基因dE 2f 1含有一个内含子microRNA簇mir 11 ~998。这种排列为miRNA与其宿主基因之间可能的功能性相互作用奠定了基础。令人惊讶的是，即使超过一半的脊椎动物miRNA是内含子，这个问题仍然在很大程度上未被认识到，因此这类miRNA很少在它们所在的基因的背景下被研究。我们最近发现mir 11 ~998簇与E2 F依赖性细胞凋亡的调节密切相关。miR-11直接调节凋亡E2 F靶点的表达以限制辐射诱导的凋亡。与miR-11不同，miR-998通过不同的机制发挥作用。在初步实验中，我们发现miR-998通过调节促生存EGFR信号传导来调节E2 F依赖性凋亡。我们将根据这些初步调查结果， 嵌入dE 2f 1基因的mir 11 ~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对果蝇中EGFR信号传导的影响。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miR-29（一种哺乳动物miR-998同源物）与EGFR信号传导之间的联系。在Aim 3中，我们将研究mir 11 ~998簇及其宿主基因dE 2f 1的共调控。我们将使用新的敲入等位基因确定簇内miR-11和miR-998之间相互依赖的功能意义。总之，这项研究将解决嵌入的miRNAs和它们的宿主基因之间的关系，并将评估Rb肿瘤抑制通路的背景下，这种相互作用的意义。值得注意的是，已知miRNA影响各种生物学过程，并在人类恶性肿瘤如癌症中发挥关键作用。因此，在成功完成该项目后产生的知识可能会刺激将miRNA调控与E2 F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.