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（细胞周期停滞、谱系特异性分化和凋亡）的生理调节剂。虽然有相当大的兴趣，丁酸作为组蛋白脱乙酰酶（HDAC）活性的抑制剂的作用，我们已经证明，丁酸触发的其他机制必须发挥重要作用，在刺激和整合的整体复杂的影响结肠细胞成熟。本申请的目的是剖析丁酸盐刺激的两种基本表观遗传机制的贡献：microRNA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，以询问改变的microRNA表达谱，以及响应丁酸盐的转录衰减的全基因组改变。至于microRNA，我们的初步数据表明，已知在低等生物以及人类癌症中协调增殖和分化的关键分子及其前体发生了变化。在更广泛的实验以确认和扩展这些数据之后，包括对1，25-二羟基维生素D3和舒林酸的比较反应，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在直接取代丁酸酯中的功能，以触发结肠细胞成熟的细胞和分子方面，或调节对丁酸酯的反应。至于转录衰减，我们发表和提交的数据是使用一种新的方法，在间期核的转录位点的成像，以证明丁酸盐导致转录衰减在降低稳态水平的c-myc和细胞周期蛋白D1基因。此外，使用我们设计和制造的新型“5 '/3'阵列”进行的全基因组扫描提供了证据，证明该机制在整个基因组的基因座上更广泛地由丁酸盐触发。这将得到证实和扩展，并在候选基因座的转录衰减的明确证据开发使用ChIP（染色质免疫沉淀）芯片上的方法。我们已发表和未发表的数据已经确定，在诱导结肠细胞成熟时，短链脂肪酸丁酸盐既改变了microRNA分子的表达，又导致许多基因的转录暂停。本申请的目标是：剖析丁酸盐对这些重新编程细胞的表观遗传机制的影响;了解它们如何与触发和整合细胞增殖、谱系特异性分化和细胞凋亡（包括细胞成熟）的途径有关;并比较和对比丁酸盐与维生素D3和非甾体抗-炎性药物舒林酸也通过这些表观遗传机制诱导成熟途径。这将使用我们开发的新方法和基因“芯片”。这些结果将确定建立肠粘膜稳态所必需的新机制，以及这些机制如何改变结肠癌的发生概率和发展。

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.