Characterization of the oncogenic potential of circRNAs in melanoma

总结 在过去的十年中，非编码RNA（ncRNA）已经成为参与转录调控的重要参与者。 调节大多数，如果不是全部，生物过程。考虑到它们的重要作用，可以想象， 有助于癌症等疾病的发展。环状RNA（CircRNA）是一种非编码RNA， 通过线性前mRNA的选择性剪接经由称为反向剪接的过程形成。circRNA是 丰富，稳定，进化保守，并显示细胞类型特异性表达模式，表明， 它们可能在细胞稳态、分化、发育和疾病中起重要作用。但 circRNA的形成，它们的调节和它们的功能知之甚少，部分原因是缺乏工具。 来驱动circRNA的永久异位表达。我们已经开发了稳定过表达的遗传工具 候选circRNA在体外和体内，我们将实施作为这个R21提案的一部分。使用这些 工具，我们将确定我们假设促进黑色素瘤发生的两种环RNA的致癌潜力。 我们通过一个综合的circRNA检测，鉴定了circPMS1和circDNAJC 2作为黑色素瘤的候选癌基因。 在良性黑素细胞和恶性黑素瘤细胞中的表达分析。我们将检测这些基因的过度表达 circDNAs促进体外黑素细胞转化和我们的高通量黑色素瘤中的肿瘤形成 小鼠模型。我们还将开始对circPMS 1的潜在功能进行分子表征， circDNAJC2致癌活性，重点是肽翻译和miRNA海绵。而且我们 将在体外以及异种移植物中特异性沉默人黑素瘤细胞中的circPMS 1和circDNAJC 2 模型来检查circRNA表达是否是维持转化状态所必需的，并继续 黑色素瘤生长。我们提出的研究将证明circPMS1或 circDNAJC 2驱动黑色素瘤的发生，并建立了一个易于适应的实验框架， 为今后的致癌circRNA研究指明了方向。

SUMMARY Over the last decade noncoding RNAs (ncRNAs) have emerged as prominent players that are involved in the regulation of most, if not all, biological processes. Given their important roles, it is conceivable that ncRNAs contribute to the development of diseases such as cancer. Circular RNAs (circRNAs) are ncRNAs that are formed through alternative splicing of linear pre-mRNAs via a process termed backsplicing. circRNAs are abundant, stable, and evolutionarily conserved and display cell-type specific expression patterns, indicating that they may play important roles in cell homeostasis, differentiation, development, and disease. However, the formation of circRNAs, their regulation, and their functions are poorly understood, partially due to the lack of tools to drive permanent ectopic expression of circRNAs. We have developed genetic tools for stable overexpression of candidate circRNAs in vitro and in vivo, which we will implement as part of this R21 proposal. Using these tools, we will determine the oncogenic potential of two circRNAs we hypothesize to promote melanomagenesis. We identified circPMS1 and circDNAJC2 as candidate melanoma oncogenes by a comprehensive circRNA expression analysis in benign melanocytes and malignant melanoma cells. We will test if overexpression of these circDNAs promotes melanocyte transformation in vitro and tumor formation in our high-throughput melanoma mouse models. We will also begin the molecular characterization of the functions underlying circPMS1 and circDNAJC2 oncogenic activity, with an emphasis on peptide translation and miRNA sponging. Moreover, we will specifically silence circPMS1 and circDNAJC2 in human melanoma cells in vitro as well as in xenograft models to examine if circRNA expression is required for maintaining the transformed state and continued melanoma growth, respectively. Our proposed studies will demonstrate that overexpression of circPMS1 or circDNAJC2 drives melanomagenesis and establish an easily adaptable experimental framework that will pave the way for future studies of oncogenic circRNAs.