FATTY ACID MODULATION OF COLON CELL TRANSFORMATION

描述（由申请人提供）：短链脂肪酸丁酸盐是结肠上皮细胞成熟（细胞周期阻滞、谱系特异性分化和凋亡）的生理调节剂。虽然人们对丁酸盐作为组蛋白去乙酰化酶（HDAC）活性抑制剂的作用相当感兴趣，但我们已经证明，丁酸盐引发的其他机制在刺激和整合结肠细胞成熟的整体复杂影响中必须发挥重要作用。本应用的目的是剖析丁酸盐刺激的两个基本表观遗传机制的贡献：microRNAs表达的改变和转录衰减。我们将首先使用我们设计和制造的新型基因表达阵列来解决这些问题，以询问丁酸盐对microRNA表达谱的改变以及转录衰减的全基因组改变。关于microrna，我们的初步数据显示了关键分子及其前体的改变，这些分子和前体在低等生物和人类癌症中协调增殖和分化。通过更广泛的实验来证实和扩展这些数据，包括对1,25-二羟基维生素D3和sulindac的比较反应，以及沿着吸收或分泌细胞谱系的自发分化，我们将确定关键microrna在直接取代丁酸盐触发结肠细胞成熟的细胞和分子方面的功能，或调节对丁酸盐的反应。关于转录衰减，我们发表和提交的数据是使用一种新的间期细胞核转录位点成像方法生成的，以证明丁酸盐导致转录衰减，降低c-myc和cyclin D1基因的稳态水平。此外，使用我们设计和制造的新型“5’/3’阵列”的全基因组扫描提供了证据，表明丁酸盐在整个基因组的位点上更广泛地触发了这种机制。这将得到证实和扩展，并使用芯片上的ChIP（染色质免疫沉淀）方法开发候选位点转录衰减的明确证据。我们已发表和未发表的数据已经证实，在诱导结肠细胞成熟过程中，短链脂肪酸丁酸盐既改变microRNA分子的表达，又导致许多基因的转录暂停。该应用程序的目标是：剖析丁酸盐对细胞重编程的这些表观遗传机制的影响；了解它们如何与细胞增殖、谱系特异性分化和细胞凋亡的触发和整合途径相关联，这些途径构成细胞成熟；并比较和对比丁酸盐与维生素D3和非甾体抗炎药sulindac通过这些表观遗传机制诱导成熟途径的影响。这将使用我们开发的新方法和基因“芯片”。这些结果将定义肠粘膜稳态建立的新机制，以及这些机制如何异常地改变结肠癌的发生和发展的可能性。

DESCRIPTION (provided by applicant): The short chain fatty acid butyrate is a physiological regulator of colon epithelial cell maturation (cell cycle arrest, lineage specific differentiation and apoptosis). While there is considerable interest in the role of butyrate as an inhibitor of histone deacetylase (HDAC) activity, we have demonstrated that other mechanisms triggered by butyrate must play essential roles in the stimulation and integration of the overall complex affects on colon cell maturation. The goal of this application is to dissect the contribution of two fundamental epigenetic mechanisms stimulated by butyrate: alteration of expression of microRNAs, and transcriptional attenuation. We will first address these issues using novel gene expression arrays that we have designed and fabricated to interrogate altered profiles of microRNA expression, and genome wide alterations in transcriptional attenuation, in response to butyrate. As regards microRNAs, our preliminary data demonstrate alterations in key molecules, and their precursors, known to coordinate proliferation and differentiation in lower organisms, as well as in human cancer. Following more extensive experiments to confirm and extend these data, including comparative responses to 1,25-dihydroxyvitamin D3 and sulindac, as well as in spontaneous differentiation along the absorptive or secretory cell lineage, we will determine the functions of key microRNAs in directly substituting for butyrate in triggering cellular and molecular aspects of colonic cell maturation, or in modulating the response to butyrate. As regards transcriptional attenuation, our published and submitted data were generated using a novel method of imaging of transcription sites in interphase nuclei to demonstrate that butyrate causes transcriptional attenuation in reducing steady state levels of the c-myc and cyclin D1 genes. Moreover, a genome wide scan using a novel "5'/3' array" that we designed and fabricated has provided evidence that this mechanism is more widely triggered by butyrate at loci throughout the genome. This will be confirmed and extended, and definitive evidence for transcriptional attenuation at candidate loci developed using a ChIP (chromatin immunoprecipitation) on chip approach. Our published and unpublished data have established that in inducing colonic cell maturation, the short chain fatty acid butyrate both alters the expression of microRNA molecules and causes transcriptional pausing at a number of genes. The goals of this application are: to dissect the affects of butyrate on these epigenetic mechanisms that reprogram the cell; to understand how they are linked to triggering and integrating pathways of cell proliferation, lineage specific differentiation, and apoptosis that comprise cell maturation; and compare and contrast the affects of butyrate with how vitamin D3 and the non-steroidal anti-inflammatory drug sulindac also induce maturation pathways through these epigenetic mechanisms. This will use novel methodologies and gene "chips" that we have developed. The results will define new mechanisms essential for the establishment of homeostasis of the intestinal mucosa, and how these are aberrant in altering probability for, and development of, colon cancer.