Microbial regulation of intestinal gene expression through microRNA

描述(由申请人提供)：结肠中的微生物群落以重要的方式与宿主相互作用，如维持屏障功能，分解和能量利用其他不可消化的食物，以及教育宿主免疫系统。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)等疾病中发挥主要作用。通过最近在与栽培无关的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理解。我们开始了解宿主通过模式识别受体(如Toll样受体)感知微生物并与其相互作用的方式，以及微生物通过调节宿主细胞增殖和分化的短链脂肪酸丁酸盐等产品与宿主相互作用的方式。我建议研究微生物通过microRNAs影响宿主基因表达的方式。MicroRNAs(MiRNAs)是一种~22个核苷酸的非编码RNA，可降低基因表达。MiRNAs在微生物对免疫和炎症细胞的作用中发挥作用。然而，微生物通过microRNAs对正常结肠中基因表达的影响知之甚少。我选择调查miRNAs是否参与肠道热休克蛋白(HSPs)的微生物调节。热休克蛋白保护肠道上皮免受高温(发烧)、炎症、饥饿和感染等应激源的侵袭。已知热休克蛋白的表达受其信使RNA的3‘非翻译区(3’UTR)调控，该区域与microRNAs已知的靶标区域相同。我假设细菌在结肠中的区域分布影响肠道上皮细胞miRNA的表达，并且miRNAs介导微生物对Hsp70的调节。我将首先确定肠道细菌是否改变了小鼠结肠上皮细胞的miRNA图谱，方法是通过比较无菌小鼠和使用微阵列喂食常规小鼠粪便的无菌小鼠的microRNA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的表达。在发现哪些microRNAs受到细菌影响后，我将确定这些microRNAs是否属于预测针对Hsp70的那一组。我还将使用定量PCR来确定在Hsp70上具有预测结合位置的miRNAs是否改变了粪便移植的无菌小鼠或暴露在粪便中的YAMC细胞的表达。我将使用microRNA模拟物和荧光素酶报告构建体来确认这些miRNAs靶向Hsp70，当感兴趣的microRNA与已被工程构建的Hsp70区域结合时，这些构建体会发出光信号。建立微生物通过调节针对Hsp70等关键调控蛋白的miRNAs来影响宿主基因表达的联系，将为进一步研究环境和基因表达与IBD和其他疾病的发病机制提供必要的基础。

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.