Characterization of the oncogenic potential of circRNAs in melanoma

摘要 在过去的十年里，非编码RNA(NcRNAs)已经成为参与 对大多数(如果不是全部)生物过程进行监管。考虑到它们的重要作用，可以想象ncRNAs 有助于癌症等疾病的发展。环状RNA(CircRNAs)是指 通过一种称为反向剪接的过程，通过线性前mRNA的选择性剪接形成。CircRNA是 丰富、稳定和进化上保守的，并显示细胞类型特异性表达模式，表明 它们可能在细胞内稳态、分化、发育和疾病中发挥重要作用。然而， 由于缺乏工具，人们对CircRNAs的形成、调控和功能知之甚少 驱动CircRNAs的永久异位表达。我们已经开发出稳定过表达的基因工具 体外和体内候选CircRNA的研究，我们将作为R21提案的一部分实施。使用这些 工具，我们将确定两个我们假设的促进黑色素瘤发生的CircRNA的致癌潜力。 我们通过一个全面的CircRNA确定了CircPMS1和CircDNAJC2是候选的黑色素瘤癌基因 良性黑素细胞和恶性黑色素瘤细胞的表达分析。我们将测试这些基因是否过度表达 CircDNA促进体外黑素细胞转化和我们高通量黑色素瘤的肿瘤形成 老鼠模型。我们还将开始CircPMS1和CircPMS1潜在功能的分子表征 CircDNAJC2致癌活性，重点是多肽翻译和miRNA海绵。此外，我们 将在体外和异种移植中特异性地沉默人黑色素瘤细胞中的CircPMS1和CircDNAJC2 检查CircRNA表达是否需要维持转换状态并继续的模型 黑色素瘤生长情况分别为。我们建议的研究将证明CircPMS1或 CircDNAJC2推动黑色素瘤的发生，并建立了一个易于适应的实验框架，将为 致癌环状核糖核酸的未来研究方向。

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.