MicroRNAs in small intestinal epithelial cell proliferation and differentiation

描述(由申请人提供)： 小肠上皮不断地进行自我更新。肠上皮细胞从细胞增殖的隐窝迁移到绒毛，在那里终末分化的吸收细胞和分泌细胞发挥作用。MicroRNAs(MiRNAs)是一类相对较新的调节RNA，对所有动物细胞类型和组织中的细胞增殖和分化都是必不可少的。MiRNAs通常通过结合靶mRNAs的3‘非翻译区(3’UTR)来抑制基因表达。我已经确定了12个在小鼠小肠绒毛和隐窝中差异表达的miRNAs。这些miRNAs可能在调节肠上皮细胞的增殖和分化中发挥作用。我的第一个目标是，将使用合成的[SIC]miRNA模拟物和抑制剂来测试这些miRNA在体外改变肠道细胞生长和成熟的能力。此外，我发现富含隐窝的miR-152被预测以KLF4为靶点，KLF4是一种在分化的肠上皮细胞中表达的转录因子。因此，在我的第二个目标中，我将确定miR-152是否通过miRNA模拟物和抑制剂在肠道细胞系中抑制KLF4的表达，以及使用从荧光素酶下游克隆KLF4的3‘非编码区的荧光素酶报告结构。此外，绒毛丰富的miR-22在小肠中高表达。通过合作，我可以使用一只miR-22(-/-)小鼠来研究miR-22在肠道中的功能。在目标3中，我将通过表达阵列在绒毛和隐窝细胞中寻找miR-22的mRNA靶点。这些数据将与电子预测相结合，以帮助识别miR-22目标。MiR-22(-/-)小鼠也将与结直肠癌模型APC(min/)小鼠杂交，以确定miR-22缺失是否会导致肿瘤。这些数据和技术将成为我的独立研究项目的基础，该项目将研究miRNA介导的基因抑制在小肠中的机制。我的导师道格拉斯·伯林博士(肠道生理学)和乔治·卡林博士(miRNA生物学)，以及我的科学顾问委员会成员，包括苏珊·亨宁博士，都是与我的提案相关的领域的专家。我通过这个奖项接受的培训将使我能够发展在生物医学研究领域所需的技能和专业知识。 与公共健康相关：这项工作将促进我们对肠道基因调控的理解。小的RNA分子已经作为治疗剂进行了测试。因此，了解对肠上皮细胞增殖和分化至关重要的miRNAs并确定其靶点有助于我们找到治疗肠道疾病和癌症等肠道疾病的新方法，并促进肠道的再生和修复。

DESCRIPTION (provided by applicant): The small intestinal epithelium is constantly undergoing self-renewal. Intestinal epithelial cells migrate from the crypts, the site of cell proliferation, to the villi, where terminally differentiated absorptive enterocytes and secretory cells function. microRNAs (miRNAs) are a relatively new class of regulatory RNAs essential for cell proliferation and differentiation in all animal cell types and tissues studied. miRNAs generally repress gene expression by binding the 3'-untranslated region (3'UTR) of target mRNAs. I have identified 12 miRNAs that are differentially expressed in villi vs. crypts of the mouse small intestine. These miRNAs may play a role in regulating proliferation and differentiation of intestinal epithelial cells. For my first Aim, sythetic [sic] miRNA mimics and inhibitors will be used to test the ability of these miRNAs to alter intestinal cell growth and maturation in vitro. Also, I found that the crypt-enriched miR-152 is predicted to target Klf4, a transcription factor expressed in differentiated intestinal epithelial cells. Therefore, in my second Aim I will determine if miR-152 represses Klf4 expression with miRNA mimics and inhibitors in intestinal cell lines, and with a luciferase reporter construct that has the 3'UTR of Klf4 cloned downstream from luciferase. Additionally, the villus-enriched miR-22 is highly expressed in the small intestine. Through a collaboration, a miR-22(-/-) mouse is available to me to study miR-22 function in the intestine. In Aim 3, I will look for mRNA targets of miR-22 in villus and crypt cells by expression array. These data will be combined with in silico predictions to help identify miR-22 targets. miR-22(-/-) mice will also be crossed with Apc(min/+) mice, a model of colorectal cancer, to determine if loss of miR-22 is tumorigenic. These data and techniques will form the basis for my independent research program to investigate mechanisms of miRNA-mediated gene repression in the small intestine. My mentors Dr. Douglas Burrin (intestinal physiology) and Dr. George Calin (miRNA biology), and the members of my scientific advisory committee, including Dr. Susan Henning, are experts in areas relevant to my proposal. The training I receive through this award will enable me to develop the skills and expertise needed for a career in biomedical research. PUBLIC HEALTH RELEVANCE: This work will advance our understanding of gene regulation in the intestine. Small RNA molecules are already being tested as therapeutic agents. Thus, knowing the miRNAs important for intestinal epithelial cell proliferation and differentiation and identifying their targets could help us find new treatments for intestinal diseases such as intestinal bowel disease and cancer, and for promoting intestinal regrowth and repair.