Sarcoma Molecular Pathology Team, Divisions of Molecular Pathology and Cancer Therapeutics, Institute of Cancer Research, Surrey, Sutton, SM2 5NG, UK.
BMC Cancer. 2018 Feb 21;18(1):217. doi: 10.1186/s12885-018-4129-8.
MYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS-02 and MYCN reported for neuroblastomas. We sought to determine a functional role for MYCNOS-01 in alveolar rhabdomyosarcoma and neuroblastoma cells and identify any associated regulatory effects between MYCN and MYCNOS-01.
MYCNOS-01, MYCNOS-02 and MYCN expression levels were assessed in alveolar rhabdomyosarcoma and neuroblastoma cell lines and tumor samples from patients using Affymetrix microarray data and quantitative RT-PCR. Following MYCNOS-01 or MYCN siRNA knockdown and MYCNOS-01 overexpression, transcript levels were assayed by quantitative RT-PCR and MYCN protein expression assessed by Western blot and immunofluorescence. Additionally, effects on cell growth, apoptosis and cell cycle profiles were determined by a metabolic assay, caspase activity and flow cytometry, respectively.
MYCNOS-01 transcript levels were generally higher in NB and RMS tumor samples and cell lines with MYCN genomic amplification. RNA interference of MYCNOS-01 expression did not alter MYCN transcript levels but decreased MYCN protein levels. Conversely, MYCN reduction increased MYCNOS-01 transcript levels, creating a negative feedback loop on MYCN protein levels. Reduction of MYCNOS-01 or MYCN expression decreased cell growth in MYCN-amplified alveolar rhabdomyosarcoma and neuroblastoma cell lines. This is consistent with MYCNOS-01-mediated regulation of MYCN contributing to the phenotype observed.
An alternative transcript of MYCNOS, MYCNOS-01, post-transcriptionally regulates MYCN levels and affects growth in MYCN-amplified rhabdomyosarcoma and neuroblastoma cells.
MYCN 在小细胞肺癌和几种儿科肿瘤中扩增，包括肺泡横纹肌肉瘤和神经母细胞瘤。MYCN 蛋白在肺泡横纹肌肉瘤和神经母细胞瘤中均发挥关键致癌作用。MYCN 反义链（MYCNOS）是位于 MYCN 反义链上的基因，编码选择性剪接的转录本，其中两个（MYCNOS-01 和 MYCNOS-02）已知在神经母细胞瘤和小细胞肺癌中表达，并且已经报道了 MYCNOS-02 与神经母细胞瘤之间的相互调节。我们试图确定 MYCNOS-01 在肺泡横纹肌肉瘤和神经母细胞瘤细胞中的功能作用，并确定 MYCN 和 MYCNOS-01 之间的任何相关调节作用。
使用 Affymetrix 微阵列数据和定量 RT-PCR 评估肺泡横纹肌肉瘤和神经母细胞瘤细胞系以及来自患者的肿瘤样本中的 MYCNOS-01、MYCNOS-02 和 MYCN 表达水平。在进行 MYCNOS-01 或 MYCN siRNA 敲低和 MYCNOS-01 过表达后，通过定量 RT-PCR 测定转录物水平，并通过 Western blot 和免疫荧光测定 MYCN 蛋白表达。此外，通过代谢测定、半胱天冬酶活性和流式细胞术分别确定对细胞生长、细胞凋亡和细胞周期谱的影响。
在具有 MYCN 基因组扩增的 NB 和 RMS 肿瘤样本和细胞系中，MYCNOS-01 转录本水平通常较高。MYCNOS-01 表达的 RNA 干扰不会改变 MYCN 转录本水平，但会降低 MYCN 蛋白水平。相反，MYCN 减少增加了 MYCNOS-01 转录本水平，在 MYCN 蛋白水平上形成了负反馈环。MYCNOS-01 或 MYCN 表达的减少降低了 MYCN 扩增的肺泡横纹肌肉瘤和神经母细胞瘤细胞系中的细胞生长。这与 MYCNOS-01 介导的对 MYCN 水平的调节一致，有助于观察到的表型。
MYCNOS 的替代转录本 MYCNOS-01 在后转录水平上调节 MYCN 水平，并影响 MYCN 扩增的横纹肌肉瘤和神经母细胞瘤细胞的生长。
