Transcriptional profiling of 2.5 dimensional MDCK tubulogenesis

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。为了实现这一目标，我将使用RNA干扰（RNAi）方法对在新的体外培养系统条件下生长的细胞进行测试，测试候选基因和可能介导小管形成的途径的作用：2.5维培养（2.5 d）的马丁-达比犬肾（MDCK）细胞。在初步实验中，我开发了一种培养方法，2.5D，可以有效地形成MDCK小管。与肝细胞生长因子诱导的三维微管形成（一种研究体外微管形成的常用模型系统）相比，2.5D模型系统显示出更多同步的发育中间体。因此，2.5D培养更适合转录谱分析。采用cDNA微阵列技术测量小管形成过程中mRNA水平的时间变化。将数据聚类，根据表达模式揭示发育中特异性基因。分类后的基因用Cytoscape进一步分析，Cytoscape是一个蛋白质网络和可视化工具，装载了人类蛋白质相互作用图。值得注意的是，在已确定的表达模式相似的基因中，有一个子集出人意料地紧密相连，这表明存在一组具有相似蛋白质功能的共调节基因。有趣的是，还有另一个连贯的功能网络，它可能代表了对小管形成至关重要的关键信号通路。然而，这些基因如何影响小管的形成仍然是未知的。为了研究这些筛选的基因在微管形成中的作用，我建议通过RNAi阻断这些基因的功能，并研究这些基因的缺失如何影响2.5D微管形成。利用共聚焦免疫荧光显微镜对RNAi引起的形态学变化进行定性和定量分析。此外，我将研究功能阻断如何影响包括细胞外信号调节激酶（ERK）和转录激活信号换能器和激活因子（STAT）在内的小管生成信号通路。在我的初步数据中发现的潜在信号通路也将被测试。分析正常小管形成的分子基础将有助于理解病理情况，如肾脏和尿路先天性异常，常染色体显性多囊肾病及相关囊性疾病。此外，小管形成的细胞过程与肿瘤发生的细胞过程有关。小管发生和肿瘤发生都需要侵袭性上皮生长的发育程序。研究微管形成的基础生物学，有助于深入了解肿瘤生物学的细胞和分子机制，特别是转移机制。

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.