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: 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
• 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

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步，但结直肠癌（CRC）仍然是美国第三大最常见的癌症。这值得考虑其他扮演重要功能角色的途径。 microRNA（miRNA）是内源性的非编码RNA，靶向mRNA用于转录抑制作用，被认为可以调节所有人类基因的30％。我们先前的工作表明，在大约50％的CRC中，针对包括C-MYC，K-RAS，IMP-1和HMGA2在内的多个mRNA的MiRNA家族成员被下调。最近的一些研究支持了新兴范式，在这种范式中，促进胚胎或成年干细胞中“干性”的相同因素可能在肿瘤发生中起关键作用，而IMP-1可能是这样的因素。 IMP-1是Let-7的直接靶标，先前已显示用于稳定C-MYC和IGF-II mRNA，但是在正常和肿瘤肠生长中，IMP-1的机理仍然难以捉摸。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损失会降低CRC细胞系的增殖和锚固无关的生长，并且高IMP-1表达与原代CRC肿瘤中的2-catenin和K-Ras激活呈正相关。 LIN28B是Let-7的内源性阻遏物，过表达LIN28B的细胞显示IMP-1的急剧增加。在当前的建议中，我们假设IMP-1是lin28b介导的LET-7下调，IMP-1是正常肠生长和结直肠肿瘤发生的关键调节剂。该假设将通过两个相互关联的特定目的进行检验。目标1是在体外表征IMP-1在正常肠生长和LIN28B/LET-7介导的结肠肿瘤发生中的作用。这将使用IMP-1，LIN28B和/或Let-7的过表达和沉默系统来实现，并在肠道干细胞报告小鼠的培养器官中实现这一目标。相同的表达系统将用于分析CRC细胞系的增殖，迁移，侵袭和独立生长以及三维（3D）器官培养物。在功能测定中添加或抑制IGF-II将测试IGF-II在IMP-1介导的肠增长中的作用。目的2是表征体内Imp-1肿瘤发生的作用。这将使用IMP-1过表达的异种移植小鼠模型以及富含肿瘤发射特性的细胞的序列移植。将分析肿瘤数，大小和形态，以及局部和循环IGF-II。 IMP-1异种移植肿瘤将使用生物发光和荧光成像进行串行监测，以进行生长，侵袭和转移，并同时进行组织学和生物分子分析，以对IGF-II或其他途径的影响。综上所述，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，并强调评估模仿Let-7或抑制IMP-1的CRC疗法的重要性。 公共卫生相关性：大肠癌（CRC）是美国第三大最常见的癌症，尽管在确定基本原因方面取得了科学的进步，但仍需要做更多的事情才能使CRC治愈。当前的提案将使用创新和协作的方法来探索在开发过程中正常的新途径，但可能在促进CRC增长方面发挥作用。这项研究的结果将为开发CRC的新疗法或生物标志物提供证据。