Neuroblastoma (NB) is the most common extracranial solid malignancy in children. Despite current aggressive treatment regimens, the prognosis for high-risk NB patients remains poor, with the survival of less than 40%. Amplification/stabilization of MYCN oncogene, in NB is associated with a high risk of recurrence. Thus, there is an urgent need for novel therapeutics. The deregulated expression of microRNA (miR) is reported in NB; nonetheless, its effect on MYCN regulation is poorly understood. First, we identified that miR-15a-5p, miR-15b-5p, and miR-16-5p (hereafter miR-15a, miR-15b or miR-16) were down-regulated in patient-derived xenografts (PDX) with high MYCN expression. MiR targeting sequences on MYCN mRNA were predicted using online databases such as TargetScan and miR database. The R2 database, containing 105 NB patients, showed an inverse correlation between MYCN mRNA and deleted in lymphocytic leukemia (DLEU) 2, a host gene of miR-15. Moreover, overexpression of miR-15a, miR-15b or miR-16 significantly reduced the levels of MYCN mRNA and N-Myc protein. Conversely, inhibiting miR dramatically enhanced MYCN mRNA and N-Myc protein levels, as well as increasing mRNA half-life in NB cells. By performing immunoprecipitation assays of argonaute-2 (Ago2), a core component of the RNA-induced silencing complex, we showed that miR-15a, miR-15b and miR-16 interact with MYCN mRNA. Luciferase reporter assays showed that miR-15a, miR-15b and miR-16 bind with 3'UTR of MYCN mRNA, resulting in MYCN suppression. Moreover, induced expression of miR-15a, miR-15b and miR-16 significantly reduced the proliferation, migration, and invasion of NB cells. Finally, transplanting miR-15a-, miR-15b- and miR-16-expressing NB cells into NSG mice repressed tumor formation and MYCN expression. These data suggest that miR-15a, miR-15b and miR-16 exert a tumor-suppressive function in NB by targeting MYCN. Therefore, these miRs could be considered as potential targets for NB treatment.

中文翻译：

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miR-15a-5p，miR-15b-5p和miR-16-5p通过直接靶向成神经细胞瘤中的MYCN来抑制肿瘤进展。

神经母细胞瘤（NB）是儿童中最常见的颅外实体恶性肿瘤。尽管目前有积极的治疗方案，但高危NB患者的预后仍然很差，存活率不到40％。NB中MYCN致癌基因的扩增/稳定与高复发风险相关。因此，迫切需要新的疗法。NB中报道了microRNA（miR）的表达失调；但是，其对MYCN调节的作用了解甚少。首先，我们确定在患者来源的异种移植物（PDX）中，miR-15a-5p，miR-15b-5p和miR-16-5p（以下称miR-15a，miR-15b或miR-16）被下调。 MYCN高表达。使用在线数据库（例如TargetScan和miR数据库）预测了MYCN mRNA上的MiR靶向序列。R2数据库包含105个NB患者，结果显示，MYCN mRNA与miR-15的宿主基因淋巴细胞白血病（DLEU）2中的缺失呈负相关。此外，miR-15a，miR-15b或miR-16的过表达显着降低了MYCN mRNA和N-Myc蛋白的水平。相反，抑制miR可以显着提高MYCN mRNA和N-Myc蛋白水平，并增加NB细胞的mRNA半衰期。通过执行argonaute-2（Ago2）（RNA诱导的沉默复合体的核心组成部分）的免疫沉淀测定，我们表明miR-15a，miR-15b和miR-16与MYCN mRNA相互作用。萤光素酶报告基因检测显示miR-15a，miR-15b和miR-16与MYCN mRNA的3'UTR结合，导致MYCN抑制。此外，miR-15a，miR-15b和miR-16的诱导表达显着降低了NB细胞的增殖，迁移和侵袭。最后，将表达miR-15a，miR-15b和miR-16的NB细胞移植到NSG小鼠中可抑制肿瘤形成和MYCN表达。这些数据表明，miR-15a，miR-15b和miR-16通过靶向MYCN在NB中发挥肿瘤抑制功能。因此，这些miRs可被视为NB治疗的潜在靶标。