Analysis of cap-dependent translational repression by microRNAs in oncogensis

描述（由申请人提供）：mRNA翻译成蛋白质必须增加以维持癌症的恶性进展。癌症中翻译机制基因的翻译增加和表达增加通常被认为是细胞转化的结果。然而，最近的一些观察对这一概念提出了质疑。首先，限速翻译起始因子eIF4E的过表达可以转化多种细胞类型。其次，microRNA（miRNA）抑制在癌症中经常减少。因为miRNA通常抑制癌基因，所以减少miRNA抑制增加癌基因表达。第三，eIF4E参与了miRNA介导的抑制，eIF4F（eIF4E、eIF4A和eIF4G的复合物）的强烈增加抑制了miRNA功能。因此，eIF4E的上调可能不是细胞转化的被动参与者，而是可能通过增加一般翻译和抑制miRNA抑制而在肿瘤发生期间发挥积极作用。 人类黑色素瘤最适合于测试上调的eIF4E在肿瘤发生中的作用。人黑色素瘤经常上调eIF4E，下调miRNA，下调miRNA相关蛋白，包括Dicer和Argonautes（Agos），因此上调miRNA靶向的癌基因。此外，我们还获得了55个黑色素瘤短期培养物（MSTC）的集合，这些培养物已通过SNP、mRNA和miRNA表达进行了注释。此外，MSTC是实验上易处理的，容易离体生长而不需要永生化，因此准确地反映了体内肿瘤生物学和遗传学操作性。 为了确定上调eIF4E在黑色素瘤发生中的作用，我们将敲低或过表达eIF4E在MSTCs和测试增殖率，细胞周期进程，逃避复制衰老，软琼脂中的集落形成，和侵袭活性。然后，我们将通过进行代谢标记和miRNA报告基因测定来确定eIF4E扰动对一般翻译、miRNA抑制或两者的影响。为了全面鉴定受eIF4E扰动影响的所有基因，我们将在eIF4E扰动之前和之后对MSTC进行微阵列分析。为了鉴定受eIF4E扰动影响的miRNA抑制的mRNA，我们将进行Ago2免疫沉淀物的微阵列分析。eIF4E的过表达应增加miRNA靶向mRNA的表达，但应减少这些mRNA的Ago2结合。我们将通过在过度表达eIF4E的MSTC中敲除eIF4E应答基因并重新测试癌症相关表型来功能性地测试eIF4E应答基因。分析肿瘤发生过程中eIF4E表达上调与非靶向和miRNA靶向mRNA翻译增加之间的联系，将阐明黑色素瘤恶性进展的基本特性，并可能发现新的治疗靶点。 公共卫生相关性：翻译必须上调，以满足肿瘤形成增加的代谢需求。这个提议中的实验挑战了翻译增加仅仅是细胞转化的结果的概念。这些研究将揭示eIF4E表达的增加如何影响所有mRNA的翻译，包括那些被microRNA选择性抑制的mRNA，以促进肿瘤发生。

DESCRIPTION (provided by applicant): Translation of mRNAs into proteins must increase to sustain the malignant progression of cancers. Increased translation and increased expression of translation machinery genes in cancers has been generally considered to be a consequence of cellular transformation. However, several recent observations challenge this concept. First, over-expression of the rate-limiting translation initiation factor eIF4E can transform multiple cell types. Second, microRNA (miRNA) repression is frequently reduced in cancers. Because miRNAs often repress oncogenes, reduced miRNA repression increases oncogene expression. Third, eIF4E has been implicated in miRNA-mediated repression and robust increases in eIF4F (a complex of eIF4E, eIF4A and eIF4G) inhibit miRNA function. Therefore, up- regulation of eIF4E may not be a passive participant in cellular transformation but may instead play an active role during oncogenesis by increasing general translation and inhibiting miRNA repression. Human melanomas are optimally suited to test the roles of up-regulated eIF4E in oncogenesis. Human melanomas frequently up-regulate eIF4E, down-regulate miRNAs, down-regulate miRNA- associated proteins including Dicer and Argonautes (Agos), and therefore up-regulate miRNA-targeted oncogenes. Additionally, we have access to a collection of 55 melanoma short-term cultures (MSTCs) that have been annotated by SNP, mRNA, and miRNA expression. Moreover, MSTCs are experimentally-tractable, grow readily ex vivo without requiring immortilization, and thus accurately reflect tumor biology and genetics operant in vivo. To define the roles of up-regulated eIF4E in melanomagenesis, we will knockdown or over-express eIF4E in MSTCs and test proliferation rates, cell-cycle progression, escape from replicative senescence, colony formation in soft agar, and invasive activity. We will then determine the effects eIF4E perturbation on general translation, miRNA repression, or both by performing metabolic labeling and miRNA reporter assays. To comprehensively identify all genes affected by eIF4E perturbation, we will perform microarray analysis of MSTCs before and after eIF4E perturbation. To identify miRNA- repressed mRNAs affected by eIF4E perturbation, we will perform microarray analysis of Ago2 immunopreciptiates. Over-expression of eIF4E should increased expression of miRNA-targeted mRNAs but should decrease Ago2 association of those mRNAs. We will functionally test eIF4E- responsive genes by knocking them down in MSTCs over-expressing eIF4E and re-testing cancer- relevant phenotypes. Dissecting the pathways that link up-regulated eIF4E expression with increased translation of untargeted and miRNA-targeted mRNAs during oncogenesis will elucidate fundamental properties of the malignant progression of melanomas and potentially uncover new therapeutic targets. PUBLIC HEALTH RELEVANCE: Translation must be up-regulated to meet the increased metabolic demands of tumor formation. Experiments in this proposal challenge the notion that increased translation is merely a consequence of cellular transformation. These studies will reveal how increased expression of eIF4E affects translation of all mRNAs, including those selectively repressed by microRNAs, to promote oncogenesis.