Microbial regulation of intestinal gene expression through microRNA

描述（由申请人提供）：结肠中庞大的微生物群落以重要的方式与宿主相互作用，例如维持屏障功能，分解和利用其他不可消化的食物的能量，以及培养宿主免疫系统。然而，宿主/微生物的相互作用是复杂的，微生物也可能在引起炎症性肠病（IBD）等疾病中发挥重要作用。通过最近在非培养技术和生物信息学方面的进展，我们对微生物群落的复杂性有了更好的了解。我们开始了解宿主通过模式识别受体（如Toll样受体）感知微生物并与微生物相互作用的方式，以及微生物通过短链脂肪酸丁酸酯（调节宿主细胞增殖和分化）等产物与宿主相互作用的方式。我建议研究微生物通过microRNA影响宿主基因表达的方式。microRNA（miRNAs）是一种约22个核苷酸的非编码RNA，可降低基因表达。miRNA在微生物对免疫和炎症细胞的作用中发挥作用。然而，很少有人知道微生物通过microRNA对正常结肠中基因表达的影响。我选择了研究微RNA是否参与肠道热休克蛋白（Hsps）的微生物调节。热休克蛋白保护肠上皮免受热（发烧）、炎症、饥饿和感染等应激因素的影响。已知热休克蛋白的表达受其信使RNA的3'非翻译区（3' UTR）调节，该区域与已知microRNA靶向的区域相同。 我推测，在结肠中的细菌的区域分布影响肠上皮细胞的miRNA表达，和miRNA介导的调节热休克蛋白70的微生物。我将首先确定肠道细菌是否改变小鼠结肠上皮中的miRNA谱，通过比较无菌小鼠中的microRNA表达与使用微阵列喂食常规小鼠粪便的无菌小鼠中的表达。我还将用小鼠粪便处理年轻成年小鼠结肠（YAMC）细胞以测量microRNA表达。在发现哪些microRNA受到细菌影响后，我将确定这些microRNA是否在预测靶向Hsp 70的组中。我还将使用定量PCR来确定在粪便移植的无菌小鼠或小鼠粪便暴露的YAMC细胞中，具有Hsp 70上的预测结合位点的miRNA是否具有改变的表达。我将使用microRNA模拟物和荧光素酶报告基因构建体来确认这些miRNAs对Hsp 70的靶向作用，当感兴趣的microRNA结合已被工程化到构建体中的Hsp 70的一个区域时，荧光素酶报告基因构建体会发出光信号。建立微生物通过调节靶向关键调控蛋白（如Hsp 70）的miRNA影响宿主基因表达的联系，将为进一步研究将环境和基因表达与IBD和其他疾病的发病机制联系起来提供必要的基础。

DESCRIPTION (provided by applicant): The expansive microbial community in the colon interacts with the host in important ways, such as maintenance of barrier function, break-down and energy utilization of otherwise non-digestible foods, and education of the host immune system. However, host/microbe interactions are complex, and microbes may also play a major role in causing conditions such as inflammatory bowel diseases (IBD). Through recent advances in cultivation-independent technologies and bioinformatics, we are gaining a much better understanding of the complexities of microbial communities. We are beginning to understand the ways that the host senses and interacts with microbes through pattern recognition receptors ( such as toll like receptors) and the ways in which microbes interact with the host via products such as the short chain fatty acid butyrate, which regulates host cell proliferation and differentiation. I propose to study the ways in which microbes affect the host's gene expression through microRNAs. microRNAs (miRNAs) are ~22 nucleotide, non-coding RNAs that decrease gene expression. miRNAs play a role in microbial actions on immune and inflammatory cells. However, little is known of the effects of microbes on gene expression in the normal colon via microRNAs. I have chosen to investigate whether miRNAs are involved in the microbial regulation of intestinal heat shock proteins (Hsps). Hsps protect the intestinal epithelium from stressors such as heat (fever), inflammation, starvation, and infection. Heat shock protein expression is known to be regulated by the 3' untranslated region (3'UTR) of its messenger RNA, the same region that microRNAs are known to target. I hypothesize that the regional distribution of the bacteria in the colon influences intestinal epithelial miRNA expression, and that miRNAs mediate the regulation of Hsp70 by microbes. I will first determine if the enteric bacteria alter miRNA profiles in the mouse colonic epithelium though comparison of the microRNA expression in germ free mice to the expression in germ free mice that have been fed the stool of conventional mice using microarrays. I will also treat young adult mouse colon (YAMC) cells with mouse stool to measure microRNA expression. After finding which microRNAs are affected by exposure to bacteria, I will determine whether these microRNAs are amongst the group predicted to target Hsp70. I will also use quantitative PCR to determine whether miRNAs with predicted binding sites on Hsp70 have altered expression in stool- transplanted germ-free mice or mouse stool exposed YAMC cells. I will confirm targeting of these miRNAs to Hsp70 with the use of microRNA mimics and luciferase reporter constructs that emit a light signal when the microRNA of interest binds a region of Hsp70 that has been engineered into the construct. Establishing the link that microbes affect host gene expression through modulation of miRNAs that target key regulatory proteins such as Hsp70 will provide the essential foundation needed for further studies linking the environment and gene expression to the pathogenesis of IBD and other diseases.