The landscape of circular RNAs in neuroblastoma I

Circular RNAs (circRNAs), a noncoding RNA class originating from alternative splicing, are highly abundant in neural tissues and were shown to regulate gene expression e.g. by interacting with microRNAs and RNA-binding proteins. Neuroblastoma is an embryonal neoplasia, which arises from undifferentiated neural crest cells. Here, we aimed to explore, whether circRNAs influence the pathogenesis of high-risk neuroblastoma. We performed whole-transcriptome sequencing of 104 primary neuroblastoma samples of all risk-groups and identified 5,203 unique circRNAs involving 2,302 genes. Candidate circRNA expression did not correlate with host gene expression, indicating independent regulatory mechanisms. circRNAs were significantly downregulated in the MYCN-amplified high-risk tumors. These findings were independently reproduced in a tetracycline-inducible MYCN-overexpression system based on a non MYCN-amplified neuroblastoma cell line, suggesting that MYCN drives this global circRNA repression. We identified the RNA helicase DHX9 as a mediator of this global suppressive effect of MYCN on circRNAs. Comparing our RNA sequencing data with other cancers and controls revealed a circRNA subset specifically upregulated in neuroblastoma that included a circRNA derived from the ARID1A tumor suppressor gene. Specific circARID1A knockdown resulted in reduced proliferation, cell numbers and viability, prompted apoptosis and induced a differentiated phenotype. Neither knockdown, nor overexpression of circARID1A influenced ARID1A mRNA and protein levels significantly. To dissect the potential mode of function, we performed a pulldown assay with subsequent mass spectrometry. We identified the RNA-binding protein KHSRP as an interaction partner that participates in the mechanism of action of circARID1A. In summary, this study highlights an important role of circRNAs in neuroblastoma biology and may establish this RNA class as a future therapeutic target and biomarker. Induction of MYCN expression in the neuroblastoma cell line SK-N-SH for 5d

环状RNA（circRNAs）是一类来源于可变剪接的非编码RNA，在神经组织中高度富集，既往研究证实其可通过与微小RNA（microRNAs）及RNA结合蛋白（RNA-binding proteins）相互作用等途径调控基因表达。神经母细胞瘤是一种起源于未分化神经嵴细胞的胚胎性肿瘤。本研究旨在探讨环状RNA是否会影响高危型神经母细胞瘤的发病机制。我们对涵盖所有风险分组的104例原发性神经母细胞瘤样本开展了全转录组测序，共鉴定出5203个独特的环状RNA，涉及2302个基因。候选环状RNA的表达水平与宿主基因表达无显著相关性，提示其存在独立的调控机制。在MYCN扩增的高危型肿瘤中，环状RNA的表达显著下调。该研究结果在基于非MYCN扩增神经母细胞瘤细胞系构建的四环素诱导型MYCN过表达系统中得到了独立验证，表明MYCN可介导这种全局性的环状RNA抑制效应。我们鉴定出RNA解旋酶DHX9是MYCN对环状RNA产生全局抑制作用的介导因子。将本研究的RNA测序数据与其他癌症及对照样本进行比较后，我们发现了一组在神经母细胞瘤中特异性上调的环状RNA子集，其中包含一个来源于ARID1A肿瘤抑制基因的环状RNA。对circARID1A进行特异性敲低可降低细胞增殖能力、细胞数量及存活率，诱导细胞凋亡，并促使细胞呈现分化表型。无论是敲低还是过表达circARID1A，均不会显著影响ARID1A的mRNA及蛋白水平。为解析其潜在的作用机制，我们开展了下拉实验并结合后续质谱分析，鉴定出RNA结合蛋白KHSRP作为circARID1A的相互作用伴侣，参与其发挥功能的分子机制。综上，本研究揭示了环状RNA在神经母细胞瘤生物学中的重要作用，有望将该类RNA开发为未来的治疗靶点与生物标志物。在神经母细胞瘤细胞系SK-N-SH中诱导MYCN表达时长为5天。