The Novel Long Noncoding RNA linc00467 Promotes Cell Survival but Is Down-Regulated by N-Myc

The worst subtype of neuroblastoma is caused by MYCN oncogene amplification and N-Myc oncoprotein over-expression. Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and tumourigenesis. While Myc oncoproteins are well-known to exert tumourigenic effects by regulating the expression of protein-coding genes and microRNAs, little is known about which lncRNAs are Myc targets and whether the Myc target lncRNAs play a role in Myc-induced oncogenesis. Here we performed differential gene expression studies using lncRNA microarray in neuroblastoma cells after transfection with control or N-Myc-specific small interfering RNA (siRNA), and identified N-Myc target lncRNAs including the novel lncRNA linc00467, the expression and function of which were completely unknown. RT-PCR, chromatin immunoprecipitation and luciferase assays showed that N-Myc suppressed linc00467 gene expression through direct binding to the linc00467 gene promoter and reducing linc00467 promoter activity. While N-Myc suppressed the expression of RD3, the protein-coding gene immediately down-stream of linc00467 gene, through direct binding to the RD3 gene promoter and reducing RD3 promoter activity, linc00467 reduced RD3 mRNA expression. Moreover, Affymetrix microarray analysis revealed that one of genes significantly up-regulated by linc00467 siRNA was the tumour suppressor gene DKK1. Importantly, knocking-down linc00467 expression with siRNA in neuroblastoma cells reduced the number of viable cells and increased the percentage of apoptotic cells, and co-transfection with DKK1 siRNA blocked the effects. These findings therefore demonstrate that N-Myc-mediated suppression of linc00467 gene transcription counterintuitively blocks N-Myc-mediated reduction in RD3 mRNA expression, and reduces neuroblastoma cell survival by inducing DKK1 expression.

神经母细胞瘤（neuroblastoma）恶性程度最高的亚型由MYCN癌基因扩增及N-Myc癌蛋白过表达所驱动。长链非编码RNA（long noncoding RNAs，lncRNAs）作为一类关键的基因表达与肿瘤发生调控因子，正逐渐成为研究热点。已知Myc癌蛋白可通过调控蛋白编码基因与微小RNA（microRNAs，miRNAs）的表达发挥促肿瘤作用，但目前对于哪些长链非编码RNA是Myc的靶标，以及这些靶标lncRNAs是否参与Myc诱导的肿瘤发生过程，仍缺乏深入认知。
本研究通过在转染对照或N-Myc特异性小干扰RNA（small interfering RNA，siRNA）的神经母细胞瘤细胞中开展lncRNA芯片差异表达分析，筛选得到包括新型lncRNA linc00467在内的N-Myc靶标lncRNAs，而此前学界对该分子的表达与功能完全未知。
逆转录聚合酶链反应（RT-PCR）、染色质免疫沉淀及荧光素酶报告实验结果显示，N-Myc可通过直接结合linc00467基因启动子并降低其启动子活性，抑制linc00467的基因表达。同时，N-Myc还可通过直接结合紧邻linc00467基因下游的蛋白编码基因RD3的启动子并降低其启动子活性，抑制RD3的表达；而linc00467则可下调RD3的mRNA水平。
此外，Affymetrix芯片分析结果显示，经linc00467 siRNA处理后显著上调的基因之一为抑癌基因DKK1。值得注意的是，在神经母细胞瘤细胞中利用siRNA敲低linc00467的表达，可减少存活细胞数量并提升凋亡细胞比例，而联合转染DKK1 siRNA则可阻断这一效应。
综上，本研究结果表明，N-Myc介导的linc00467基因转录抑制会反常地抵消N-Myc对RD3 mRNA表达的抑制作用，并通过诱导DKK1的表达降低神经母细胞瘤细胞的存活能力。