Role of IMP-1 and let-7 miRNAs in normal intestinal growth and colorectal cancer

IMP-1 和 let-7 miRNA 在正常肠道生长和结直肠癌中的作用

基本信息

批准号：
8201847
负责人：
Kathryn Elizabeth Hamilton
金额：
$ 4.84万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8201847
关键词：
Adult Anchorage-Independent Growth Biological Assay Biological Markers Biology CD44 gene Cancer Biology Cancer Patient Cancer cell line Cell Line Cell Maintenance Cell Transplantation Cells Clinical Colon Colorectal Colorectal Cancer Colorectal Neoplasms Data Development Diagnostic Down-Regulation Dwarfism Embryo Epidermal Growth Factor Receptor Epithelial Epithelial Cells Event Exhibits Family Functional RNA Genes Growth Growth and Development function HMGA2 gene Homeostasis Human Image In Vitro Insulin-Like Growth Factor II Intestines Knowledge Luciferases MADH4 gene Malignant Neoplasms Mediating Messenger RNA MicroRNAs Migration Assay Molecular Monitor Morphology Mus Neoplasm Metastasis Oncogenes Organ Organoids Outcome Pathway interactions Peptide Initiation Factors Perinatal mortality demographics Play Population Primary Neoplasm Process Property RNA-Binding Proteins Reporter Role Scientific Advances and Accomplishments Small Intestines Somatic Cell Stem cells System Testing Therapeutic Translations Tumor Stem Cells Tumor Suppressor Genes Tumor Suppressor Proteins United States Up-Regulation Work Xenograft procedure adult stem cell c-myc Genes cancer cell cell growth colon cancer cell line gene repression in vivo innovation insight knock-down member migration mouse model neoplastic pluripotency research study stemness therapeutic target tumor tumor growth tumor initiation tumor progression tumor xenograft tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Despite advances in determining the molecular mechanisms regulating both normal and aberrant intestinal growth, colorectal cancer (CRC) remains the 3rd most common cancer in the United States. This merits consideration of other pathways that play important functional roles. MicroRNAs (miRNAs) are endogenous, non-coding RNAs that target mRNAs for transcriptional repression, and are thought to regulate up to 30% of all human genes. Our prior work revealed that members of the let-7 family of miRNAs, which target several mRNAs including c-Myc, K-ras, IMP-1 and HMGA2, are down-regulated in approximately 50% of CRCs. Several recent studies support the emerging paradigm in which the same factors that promote "stemness" in embryonic or adult stem cells may play a critical role in tumorigenesis, and IMP-1 may be such a factor. IMP-1 is a direct target of let-7 and has been shown previously to stabilize c-Myc and IGF-II mRNA, but the mechanisms of IMP-1 in normal and neoplastic intestinal growth remain elusive. Prior studies suggest IMP-1 may have a role in stem cell maintenance or cellular differentiation. Our preliminary data demonstrate that IMP-1 loss decreases proliferation and anchorage-independent growth of CRC cell lines, and that high IMP-1 expression is positively correlated with 2-catenin and K-ras activation in primary CRC tumors. Lin28b is an endogenous repressor of let-7, and cells over-expressing Lin28b display a dramatic increase in IMP-1. In the current proposal, we hypothesize that IMP-1 is a critical regulator of normal intestinal growth and colorectal tumorigenesis as a result of Lin28b-mediated down-regulation of let-7. This hypothesis will be tested through two interrelated specific aims. Aim 1 is to characterize the role of IMP-1 in normal intestinal growth and Lin28b/let-7-mediated colon tumorigenesis in vitro. This will be achieved using IMP-1, Lin28b, and/or let-7 over-expression and silencing systems in cultured organoids from intestinal stem cell reporter mice. The same expression systems will be used to assay proliferation, migration, invasion, and anchorage- independent growth, as well as three-dimensional (3D) organotypic cultures of CRC cell lines. Adding or inhibiting IGF-II in functional assays will test the role of IGF-II in IMP-1-mediated intestinal growth. Aim 2 is to characterize the role of IMP-1 tumorigenesis in vivo. This will be pursued using xenograft mouse models of IMP-1 over-expression, as well as serial transplantation of cells enriched for tumor-initiating properties. Tumor number, size, and morphology will be analyzed, as well as local and circulating IGF-II. IMP-1 xenograft tumors will be monitored serially for growth, invasion, and metastases using bioluminescent and fluorescent imaging, with concurrent histological and biomolecular analysis for effects on IGF-II or other pathways. Taken together, these studies will reveal mechanisms of IMP-1 in normal intestinal epithelial and CRC biology. In addition, the proposed studies will further support the tumor suppressor role of let-7 and underscore the importance of evaluating the CRC therapeutics that mimic the actions of let-7 or inhibit IMP-1. PUBLIC HEALTH RELEVANCE: Colorectal cancer (CRC) is the 3rd most common cancer in the United States, and despite scientific advances in determining the underlying causes, more needs to be done to make CRC curable. The current proposal will use innovative and collaborative approaches to explore a new pathway that is normal during development, but may play a role in promoting CRC growth. Findings from this study will provide evidence for the development of new therapies or biomarkers for CRC.

描述（由申请人提供）：尽管在确定调节正常和异常肠道生长的分子机制方面取得了进展，结直肠癌（CRC）仍然是美国第三大常见癌症。这值得考虑发挥重要功能作用的其他途径。 MicroRNA (miRNA) 是内源性非编码 RNA，以 mRNA 为靶点进行转录抑制，被认为可调节高达 30% 的人类基因。我们之前的工作表明，let-7 miRNA 家族的成员（其靶向包括 c-Myc、K-ras、IMP-1 和 HMGA2 在内的多种 mRNA）在大约 50% 的 CRC 中下调。最近的几项研究支持新兴的范式，其中促进胚胎或成体干细胞“干性”的相同因素可能在肿瘤发生中发挥关键作用，而 IMP-1 可能就是这样一个因素。 IMP-1 是 let-7 的直接靶标，之前已被证明可以稳定 c-Myc 和 IGF-II mRNA，但 IMP-1 在正常和肿瘤性肠道生长中的机制仍不清楚。先前的研究表明 IMP-1 可能在干细胞维持或细胞分化中发挥作用。我们的初步数据表明，IMP-1 缺失会降低 CRC 细胞系的增殖和贴壁依赖性生长，并且 IMP-1 高表达与原发性 CRC 肿瘤中的 2-catenin 和 K-ras 激活呈正相关。 Lin28b 是 let-7 的内源性阻遏蛋白，过度表达 Lin28b 的细胞显示 IMP-1 显着增加。在当前的提议中，我们假设 IMP-1 是正常肠道生长和结直肠肿瘤发生的关键调节因子，这是 Lin28b 介导的 let-7 下调的结果。这一假设将通过两个相互关联的具体目标进行检验。目标 1 是在体外表征 IMP-1 在正常肠道生长和 Lin28b/let-7 介导的结肠肿瘤发生中的作用。这将通过在肠道干细胞报告小鼠培养的类器官中使用 IMP-1、Lin28b 和/或 let-7 过表达和沉默系统来实现。相同的表达系统将用于测定 CRC 细胞系的增殖、迁移、侵袭和贴壁独立生长以及三维 (3D) 器官型培养物。在功能测定中添加或抑制 IGF-II 将测试 IGF-II 在 IMP-1 介导的肠道生长中的作用。目标 2 是表征 IMP-1 体内肿瘤发生的作用。这将通过使用 IMP-1 过度表达的异种移植小鼠模型以及富含肿瘤起始特性的细胞的连续移植来实现。将分析肿瘤的数量、大小和形态，以及局部和循环 IGF-II。将使用生物发光和荧光成像连续监测 IMP-1 异种移植肿瘤的生长、侵袭和转移，同时进行组织学和生物分子分析以了解对 IGF-II 或其他途径的影响。总而言之，这些研究将揭示 IMP-1 在正常肠上皮和 CRC 生物学中的机制。此外，拟议的研究将进一步支持let-7的肿瘤抑制作用，并强调评估模拟let-7或抑制IMP-1作用的CRC疗法的重要性。公共健康相关性：结直肠癌 (CRC) 是美国第三大常见癌症，尽管在确定根本原因方面取得了科学进步，但要治愈结直肠癌，还需要做更多的工作。目前的提案将使用创新和协作的方法来探索一条在开发过程中正常的新途径，但可能会在促进 CRC 增长方面发挥作用。这项研究的结果将为结直肠癌新疗法或生物标志物的开发提供证据。