Transcriptional profiling of 2.5 dimensional MDCK tubulogenesis

2.5 维 MDCK 管发生的转录谱

• 批准号: 8049634
• 负责人: Kenneth Kwon
• 金额: $ 5.58万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8049634
• 关键词: Affect; Autosomal Dominant Polycystic Kidney; Biological Models; Biological Process; Biology; Cancer Biology; Candidate Disease Gene; Canis familiaris; Cell Proliferation; Cell physiology; Cells; Cluster Analysis; Complex; Data; Development; Dimensions; Disease; Epithelial; Extracellular Signal Regulated Kinases; Focal Adhesion Kinase 1; Genes; Goals; Growth; Hepatocyte Growth Factor; Human; Imagery; Immunofluorescence Microscopy; In Vitro; Individual; Investigation; Kidney; Lead; Measures; Mediating; Messenger RNA; Methods; Microarray Analysis; Mitogen-Activated Protein Kinases; Molecular; Neoplasm Metastasis; Organ; Pathway Analysis; Pathway interactions; Pattern; Phase; Process; Protein-Protein Interaction Map; Proteins; Protocols documentation; RNA Interference; Research Proposals; Role; STAT protein; Shapes; Signal Pathway; Signal Transduction; System; Testing; Time; Transcriptional Activation; Tube; Urinary tract; base; cDNA Arrays; gene function; kidney cell; knock-down; migration; programs; protein function; research study; text searching; tool; tumorigenesis

DESCRIPTION (provided by applicant): The goal of this research proposal is to identify the genes required for tubule formation. To accomplish this, I will test the role of candidate genes and pathways that may mediate tubulogenesis by using RNA interference (RNAi) methods on cells grown under the conditions of a new in vitro culture system: 2.5 dimensional culture (2.5D) of Mardin-Darby canine kidney (MDCK) cells. In preliminary experiments, I developed a culture method, 2.5D, that allows efficient MDCK tubulogenesis. Compared to hepatocyte growth factor-induced three-dimensional tubulogenesis, a common model system for investigation of in vitro tubulogenesis, the 2.5D model system shows more synchronized developmental intermediates. Hence 2.5D cultures are better suited for transcriptional profiling analysis. cDNA microarrays were used to measure the temporal changes of mRNA levels during tubulogenesis. The data were clustered to reveal developmental intermediate-specific genes on the basis of expression pattern. Classified genes were further analyzed with Cytoscape, a protein network and visualization tool loaded with the human protein interaction map. Notably, one subset of the identified genes whose expression patterns are similar is surprisingly well connected, indicating the existence of a group of co-regulated genes with similar protein functions. Interestingly, there is another coherent functional network, which may represent a key signaling pathway critical to tubulogenesis. However, it is still unknown how these genes affect tubule formation. To investigate the roles of these screened genes in tubulogenesis, I propose to block the function of the genes by RNAi, and examine how the depletion affects 2.5D tubulogenesis. Using confocal immunofluorescence microscopy, morphological changes caused by the RNAi will be analyzed qualitatively and quantitatively. In addition, I will examine how the functional blockade affects tubulogenic signaling pathways including extracellular signal regulated kinase (ERK) and signal transducer and activator of transcription activation (STAT). The potential signaling pathways found in my preliminary data will also be tested. Analyzing the molecular basis for normal tubulogenesis will contribute to understanding pathological conditions, such as congenital anomalies of the kidney and urinary tract, and autosomal dominant polycystic kidney disease and related cystic diseases. In addition, the cellular processes in tubulogenesis are related to those in tumorigenesis. Both tubulogenesis and tumorigenesis require a developmental program of invasive epithelial growth. Studying the basic biology of tubulogenesis may lead to a deeper understanding of the cellular and molecular mechanisms of cancer biology, and in particular metastasis.

描述（由申请人提供）：本研究计划的目标是确定小管形成所需的基因。为了实现这一点，我将测试候选基因和途径的作用，可能介导的小管通过使用RNA干扰（RNAi）的方法对细胞生长的条件下，一个新的体外培养系统：2.5维培养（2.5D）的Mardin-Darby犬肾（MDCK）细胞。在初步实验中，我开发了一种培养方法，2.5D，允许有效的MDCK微管发生。与肝细胞生长因子诱导的三维小管形成（一种用于体外小管形成研究的常见模型系统）相比，2.5D模型系统显示出更多的同步发育中间体。因此，2.5D培养物更适合于转录谱分析。cDNA微阵列被用来测量mRNA水平的时间变化过程中小管。对数据进行聚类，以揭示基于表达模式的发育中间体特异性基因。分类的基因进一步分析与Cytoscape，蛋白质网络和可视化工具加载与人类蛋白质相互作用的地图。值得注意的是，一个子集的鉴定基因的表达模式是相似的是令人惊讶的良好连接，表明存在一组具有相似的蛋白质功能的共调控基因。有趣的是，还有另一个连贯的功能网络，这可能是一个关键的信号通路至关重要的小管。然而，这些基因如何影响小管形成仍然是未知的。为了研究这些筛选出的基因在微管发生中的作用，我建议通过RNAi来阻断这些基因的功能，并研究缺失如何影响2.5D微管发生。使用共聚焦免疫荧光显微镜，定性和定量分析RNAi引起的形态学变化。此外，我将研究功能性阻断如何影响微管发生信号通路，包括细胞外信号调节激酶（ERK）和信号转导和转录激活因子（STAT）。在我的初步数据中发现的潜在信号通路也将被测试。分析正常肾小管形成的分子基础将有助于了解病理条件，如先天性肾脏和泌尿道异常，常染色体显性遗传性多囊肾病和相关的囊性疾病。此外，小管形成的细胞过程与肿瘤发生的细胞过程相关。小管发生和肿瘤发生都需要侵入性上皮生长的发育程序。研究微管发生的基础生物学可能会导致对癌症生物学的细胞和分子机制的更深入理解，特别是转移。