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

• 批准号: 8321674
• 负责人: Kathryn Elizabeth Hamilton
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321674
• 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

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.

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，但结直肠癌（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（miRNAs）是内源性的非编码RNA，其靶向mRNA进行转录抑制，并且被认为调节高达30%的所有人类基因。我们先前的工作显示，靶向包括c-Myc、K-ras、IMP-1和HMGA 2在内的几种mRNA的let-7家族的miRNA成员在大约50%的CRC中下调。最近的几项研究支持新出现的范式，其中促进胚胎或成体干细胞中的“干细胞性”的相同因子可能在肿瘤发生中起关键作用，IMP-1可能是这样一个因子。IMP-1是let-7的直接靶点，并且先前已经显示出稳定c-Myc和IGF-II mRNA，但是IMP-1在正常和肿瘤性肠生长中的机制仍然不清楚。先前的研究表明IMP-1可能在干细胞维持或细胞分化中起作用。我们的初步数据表明，IMP-1的损失降低CRC细胞系的增殖和锚定非依赖性生长，并且IMP-1的高表达与原发性CRC肿瘤中的2-catenin和K-ras活化正相关。Lin 28 b是let-7的内源性阻遏物，并且过表达Lin 28 b的细胞显示IMP-1的显著增加。在目前的提议中，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Lin 28 b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。目的1：研究IMP-1在正常肠道生长和Lin 28 b/let-7介导的结肠肿瘤发生中的作用。这将在来自肠干细胞报告小鼠的培养的类器官中使用IMP-1、Lin 28 b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、侵袭和锚定非依赖性生长以及三维（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。目的2是表征IMP-1在体内肿瘤发生中的作用。这将使用IMP-1过表达的异种移植小鼠模型以及富集肿瘤引发特性的细胞的连续移植来进行。将分析肿瘤数量、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，以确定对IGF-II或其他途径的影响。总之，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作用或抑制IMP-1的CRC治疗的重要性。