Control of Proliferation-related Genes and miRs by Nkx2-1 in Lung Development

项目摘要/摘要 此应用程序中包含的研究和培训计划旨在提高我的 在肺细胞和分子生物学方面的专业知识，并发展在肺脏从事独立职业的技能 研究。通过这个KO1项目，我希望提高我在项目设计、拨款撰写、项目和 实验室管理层要及时成功地过渡到独立的调查员。我的近期目标是 接受高级培训，分析肺基因调控的转录机制，以及 复杂的肺表型。 在我的导师和共同导师，这些领域的专家的支持下，我建议分析 Nkx2-1对增殖相关基因的调控及其对肺上皮细胞增殖的影响 Nkx2-1突变小鼠。Nkx2-1转录因子对肺分支和远端上皮细胞是必不可少的 发育过程中的细胞分化。在成人中，Nkx2-1通过细胞维持远端上皮表型 特定的基因调控。在肺肿瘤中，Nkx2-1是一种谱系生存癌基因，是一种预后因素。 Nkx2-1在控制细胞增殖中的作用已被提出，但可能介导细胞增殖的靶基因 在体内的增殖是未知的。该项目的目标是评估Nkx2-1直接靶标的作用 Nkx2-1在小鼠肺发育和疾病中调控细胞增殖的基因。 我们之前的芯片分析显示，与增殖相关的基因和 肺发育早期Nkx2-1结合的miRNAs数目。这里我假设Nkx2-1 通过这些基因和miRNAs调节肺内细胞的增殖，它们对 Nkx2-1功能异常改变时肺表型异常的发病机制。 这一假设将通过以下方式得到验证：1)分析选定细胞的发育表达 之前在我们的屏幕上发现的与增殖相关的Nkx2-1靶标，并评估Nkx2-1与 2)Nkx2-1调控的miRNAs及其mRNAs的表达特征 与体外细胞增殖相关，并将它们的表达与下游基因的表达相关联，以建立 Nkx2-1驱动的miRNA调控网络参与肺细胞增殖；3)评估 Nkx2-1靶基因和miRNAs在Nkx2-1缺失突变小鼠肺组织表型中的作用 在Nkx2-1磷酸化突变体中，哪些分支形态发生和分化受到破坏 尽管肺分支正常，但肺发育不良的肺。这些将为我们提供对 磷酸化、非磷酸化和缺失Nkx2-1在细胞增殖控制中的不同作用 体内的基因。 波士顿大学医学院的肺中心因其 支持环境，帮助初级教师向独立的职业生涯过渡。周刊 与我的导师和共同导师的讨论，正在进行的工作会议和教职员工 医学部提供的发展计划是我培训计划的关键组成部分。

PROJECT SUMMARY/ABSTRACT The research and training plans included in this application are designed to increase my expertise in lung cell and molecular biology, and to develop skills to pursue an independent career in lung research. With this KO1 project I expect to advance my skills in project design, grant writing, project and lab management to succeed in a timely transition to independent investigator. My immediate goals are to acquire advanced training in the analysis of transcriptional mechanisms of lung gene regulation, and of complex lung phenotypes. With the support of my mentor and co-mentors, experts in these fields, I propose to analyze Nkx2-1 regulation of proliferation-related genes and their effect on lung epithelial cell proliferation in Nkx2-1 mutant mice. The Nkx2-1 transcription factor is essential for lung branching and distal epithelial cell differentiation during development. In the adult, Nkx2-1 maintains distal epithelial phenotypes by cell specific gene regulation. In lung tumors, Nkx2-1 is a lineage survival oncogene and a prognostic factor. A role of Nkx2-1 in control of cell proliferation has been suggested, but the target genes that may mediate proliferation in vivo are unknown. The goal of this project is to evaluate the role of Nkx2-1 direct target genes in the regulation of cell proliferation attributed to Nkx2-1 in mouse lung development and disease. Our previous ChIP-on-chip analyses revealed an overrepresentation of proliferation-related genes and a number of miRNAs bound by Nkx2-1 in early lung development. Here I hypothesize that Nkx2-1 regulates cell proliferation in the lung through these genes and miRNAs, and that they contribute to the pathogenesis of the abnormal lung phenotypes when altered by disruption of Nkx2-1 function. This hypothesis will be tested by: 1) Analyzing the developmental expression of selected cell proliferation-related Nkx2-1 targets previously identified in our screen, and evaluating Nkx2-1 binding to these targets in lung tissues, 2) Characterizing expression of Nkx2-1-regulated miRNAs and their mRNAs linked to cell proliferation in vitro and correlate their expression to that of downstream genes to establish an Nkx2-1 driven miRNA regulatory network involved in lung cell proliferation; 3) Evaluating the contribution of these Nkx2-1 target genes and miRNAs to the phenotype of Nkx2-1 null mutant lungs in which branching morphogenesis and differentiation are disrupted, and in Nkx2-1 phosphorylation mutant lungs in which the lung is hypoplastic in spite of normal branching. These will provide insights into the differential role of phosphorylated, unphosphorylated or absent Nkx2-1 in control of cell proliferation genes in vivo. The Pulmonary Center at Boston University School of Medicine is widely recognized for its supporting environment, helping Junior Faculty to transition to an independent career. The weekly discussions with my mentor and co-mentors, the work-in-progress meetings and the Faculty Development Program offered by the Department of Medicine are key components of my training plan.