Transcriptional profiling of 2.5 dimensional MDCK tubulogenesis

描述(由申请人提供)：这项研究计划的目标是确定小管形成所需的基因。为此，我将通过使用RNA干扰(RNAi)方法，在一种新的体外培养系统下生长的细胞上测试可能介导小管形成的候选基因和途径的作用：2.5D的Mardin-Darby犬肾(MDCK)细胞。在初步实验中，我开发了一种2.5D的培养方法，允许高效的MDCK小管形成。与肝细胞生长因子诱导的三维小管发生相比，2.5D模型系统显示了更多的同步发育中间产物。因此，2.5D培养更适合于转录图谱分析。用基因芯片技术检测肾小管发生过程中基因表达水平的变化。根据表达模式对数据进行聚类，揭示发育中间特异型基因。利用加载了人类蛋白质相互作用图的蛋白质网络和可视化工具Cytoscape对分类基因进行进一步分析。值得注意的是，表达模式相似的已识别基因中的一个子集惊人地紧密相连，这表明存在一组具有相似蛋白质功能的共同调控基因。有趣的是，还有另一个连贯的功能网络，它可能代表着对小管形成至关重要的关键信号通路。然而，目前还不清楚这些基因是如何影响小管形成的。为了研究这些筛选的基因在小管发生中的作用，我建议用RNAi来阻断这些基因的功能，并研究这种缺失如何影响2.5D小管发生。利用共聚焦免疫荧光显微镜，将定性和定量地分析RNAi引起的形态变化。此外，我将研究功能阻断如何影响小管生成信号通路，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。分析正常肾小管发生的分子基础有助于了解肾脏和尿路的先天性异常、常染色体显性遗传性多囊肾病及相关囊性疾病等病理情况。此外，小管形成过程中的细胞过程与肿瘤发生过程中的细胞过程有关。肾小管的形成和肿瘤的发生都需要一个侵袭性上皮生长的发育程序。研究小管发生的基础生物学有助于更深入地了解肿瘤生物学，特别是转移的细胞和分子机制。

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.