Zhao X, Li D, Pu J, Mei H, Yang D, Xiang X, Qu H, Huang K, Zheng L, Tong Q
Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China.
Clinical Center of Human Genomic Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China.
Oncogene. 2016 Jul 7;35(27):3565-76. doi: 10.1038/onc.2015.422. Epub 2015 Nov 9.
Previous studies have indicated the important roles of MYCN in tumorigenesis and progression of neuroblastoma (NB), the most common extracranial solid tumor derived from neural crest in childhood. However, the regulatory mechanisms of MYCN expression in NB still remain largely unknown. In this study, through mining public microarray databases and analyzing the cis-regulatory elements and chromatin immunoprecipitation data sets, we identified CCCTC-binding factor (CTCF) as a crucial transcription factor facilitating the MYCN expression in NB. RNA immunoprecipitation, RNA electrophoretic mobility shift assay, RNA pull down and in vitro binding assay indicated the physical interaction between CTCF and MYCN opposite strand (MYCNOS), a natural noncoding RNA surrounding the MYNC promoter. Gain- and loss-of-function studies revealed that MYCNOS facilitated the recruitment of CTCF to its binding sites within the MYCN promoter to induce chromatin remodeling, resulting in enhanced MYCN levels and altered downstream gene expression, in cultured NB cell lines. CTCF cooperated with MYCNOS to suppress the differentiation and promote the growth, invasion and metastasis of NB cells in vitro and in vivo. In clinical NB tissues and cell lines, CTCF and MYCNOS were upregulated and positively correlated with MYCN expression. CTCF was an independent prognostic factor for unfavorable outcome of NB, and patients with high MYCNOS expression had lower survival probability. Taken together, these results demonstrate that CTCF cooperates with noncoding RNA MYCNOS to exhibit oncogenic activity that affects the aggressiveness and progression of NB through transcriptional upregulation of MYCN.
先前的研究表明，MYCN在神经母细胞瘤（NB）的肿瘤发生和进展中发挥重要作用，NB是儿童期最常见的源自神经嵴的颅外实体瘤。然而，NB中MYCN表达的调控机制仍 largely未知。在本研究中，通过挖掘公共微阵列数据库并分析顺式调控元件和染色质免疫沉淀数据集，我们鉴定出CCCTC结合因子（CTCF）是促进NB中MYCN表达的关键转录因子。RNA免疫沉淀、RNA电泳迁移率变动分析、RNA下拉和体外结合分析表明CTCF与MYCN反义链（MYCNOS）存在物理相互作用，MYCNOS是围绕MYNC启动子的天然非编码RNA。功能获得和功能缺失研究表明，在培养的NB细胞系中，MYCNOS促进CTCF募集至MYCN启动子内其结合位点以诱导染色质重塑，导致MYCN水平升高并改变下游基因表达。CTCF与MYCNOS协同作用以在体外和体内抑制NB细胞分化并促进其生长、侵袭和转移。在临床NB组织和细胞系中，CTCF和MYCNOS上调且与MYCN表达呈正相关。CTCF是NB不良预后的独立预后因素，MYCNOS高表达的患者生存概率较低。综上所述，这些结果表明CTCF与非编码RNA MYCNOS协同发挥致癌活性，通过转录上调MYCN影响NB的侵袭性和进展。
