Analysis of cap-dependent translational repression by microRNAs in oncogensis

描述(由申请人提供)：必须增加mRNAs到蛋白质的翻译，以维持癌症的恶性进展。在癌症中，翻译机器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，最近的一些观察结果对这一概念提出了挑战。首先，限速翻译起始因子eIF4E的过度表达可以转化多种细胞类型。其次，在癌症中，microRNA(MiRNA)的抑制作用经常降低。因为miRNA经常抑制癌基因，所以miRNA抑制减少会增加癌基因的表达。第三，eIF4E与miRNA介导的抑制有关，eIF4F(eIF4E、eIF4A和eIF4G的复合体)的强劲增加抑制了miRNA的功能。因此，eIF4E的上调可能不是细胞转化的被动参与者，而是通过增加一般翻译和抑制miRNA抑制而在肿瘤发生过程中发挥积极作用。人类黑色素瘤最适合测试上调的eIF4E在肿瘤发生中的作用。人类黑色素瘤经常上调eIF4E，下调miRNAs，下调miRNA相关蛋白，包括DICER和ArgAertes(AGOS)，从而上调miRNA靶向的癌基因。此外，我们还可以访问55个黑色素瘤短期培养细胞(MSTC)的集合，这些细胞已经被SNP、mRNA和miRNA表达注释。此外，MSTCs在实验上容易驯化，在体外很容易生长，不需要永生化，从而准确地反映了体内的肿瘤生物学和遗传学操作。为了确定上调的eIF4E在黑色素瘤发生中的作用，我们将在MSTCs中敲除或过表达eIF4E，并测试增殖率、细胞周期进展、逃避复制衰老、软琼脂中的克隆形成和侵袭活性。然后，我们将通过进行代谢标记和miRNA报告程序分析，确定eIF4E扰动对一般翻译、miRNA抑制或两者的影响。为了全面识别所有受eIF4E干扰影响的基因，我们将对eIF4E干扰前后的MSTC进行微阵列分析。为了识别受eIF4E干扰影响的miRNA抑制的mRNAs，我们将对Ago2免疫沉淀物进行微阵列分析。EIF4E的过表达应该增加miRNA靶向的mRNAs的表达，但应该减少这些mRNAs的Ago2结合。我们将通过在过度表达eIF4E的MSTC中敲除eIF4E反应基因并重新测试与癌症相关的表型来从功能上测试eIF4E反应基因。剖析在肿瘤发生过程中上调eIF4E表达与非靶向和miRNA靶向mRNAs翻译增加之间的联系的途径将阐明黑色素瘤恶性进展的基本特征，并可能发现新的治疗靶点。

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.