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

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。这值得考虑其他发挥重要功能作用的途径。MicroRNAs （miRNAs）是内源性的非编码rna，以mrna为目标进行转录抑制，被认为可以调节多达30%的人类基因。我们之前的工作表明，let-7 mirna家族的成员在大约50%的crc中下调，其靶向多种mrna，包括c-Myc、K-ras、IMP-1和HMGA2。最近的一些研究支持了新兴的范式，即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起关键作用，而IMP-1可能就是这样一个因素。IMP-1是let-7的直接靶点，先前已被证明可以稳定c-Myc和IGF-II mRNA，但IMP-1在正常和肿瘤肠道生长中的机制尚不清楚。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的缺失会降低CRC细胞系的增殖和不依赖锚定的生长，并且在原发性CRC肿瘤中，IMP-1的高表达与2-catenin和K-ras的激活呈正相关。Lin28b是let-7的内源性抑制因子，过表达Lin28b的细胞表现出IMP-1的急剧增加。在目前的提案中，我们假设由于lin28b介导的let-7下调，IMP-1是正常肠道生长和结直肠肿瘤发生的关键调节因子。这一假设将通过两个相互关联的具体目标来检验。目的1是表征IMP-1在体外正常肠道生长和Lin28b/let-7介导的结肠肿瘤发生中的作用。这将通过在培养的肠干细胞报告小鼠类器官中使用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检测CRC细胞系的增殖、迁移、侵袭和非锚定生长，以及三维（3D）器官型培养。在功能分析中添加或抑制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。这将通过使用IMP-1过表达的异种移植小鼠模型，以及具有肿瘤启动特性的富集细胞的连续移植来实现。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对IMP-1异种移植肿瘤的生长、侵袭和转移进行连续监测，同时对其对IGF-II或其他途径的影响进行组织学和生物分子分析。综上所述，这些研究将揭示IMP-1在正常肠上皮和结直肠癌生物学中的作用机制。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作用的结直肠癌治疗方法的重要性。

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.