FATTY ACID MODULATION OF COLON CELL TRANSFORMATION

描述(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。虽然丁酸作为组蛋白脱乙酰基酶(HDAC)活性抑制剂的作用引起了相当大的兴趣，但我们已经证明，丁酸触发的其他机制肯定在刺激和整合整个复杂的影响结肠细胞成熟的过程中发挥重要作用。本申请的目的是剖析丁酸刺激的两种基本表观遗传机制的贡献：microRNAs表达的改变和转录减弱。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，这些阵列用于询问改变的microRNA表达谱，以及全基因组范围内转录衰减的变化，以响应丁酸盐。至于microRNAs，我们的初步数据显示了关键分子及其前体的变化，这些分子和前体已知在低等生物中协调增殖和分化，在人类癌症中也是如此。在更广泛的实验来证实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，以及在吸收或分泌细胞谱系的自发分化中，我们将确定关键的microRNAs在直接替代丁酸在触发结肠细胞成熟的细胞和分子方面，或在调节对丁酸的反应方面的功能。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核转录位点成像方法产生的，以证明丁酸盐通过降低c-myc和cyClinD1基因的稳定水平而导致转录衰减。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，表明这种机制更广泛地由整个基因组中的丁酸盐基因座触发。这将得到证实和扩展，并确定使用芯片(染色质免疫沉淀)芯片方法开发的候选基因座转录减弱的确凿证据。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的过程中，短链脂肪酸丁酸盐既改变了microRNA分子的表达，又导致了一些基因的转录暂停。这项应用的目标是：剖析丁酸盐对这些对细胞重新编程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成熟的凋亡途径相联系；以及比较和对比丁酸与维生素D3和非类固醇抗炎药物舒林酸如何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。这一结果将确定建立肠粘膜内稳态所必需的新机制，以及这些机制在改变结肠癌发生和发展的可能性方面是如何异常的。

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.