Transcriptomic analysis of neuroblastoma cells in response to stable over-expression of myeloid zinc finger 1 antisense RNA 1 (MZF1-AS1)

Neuroblastoma (NB), a malignant embryonic tumor arising from primitive neural crest cells, accounts for more than 7% of malignancies and around 15% of cancer-related mortality in childhood. Better elucidating the mechanisms of tumorigenesis and aggressiveness is important for improving the therapeutic efficiencies of NB. Through mining of public datasets, we identified myeloid zinc finger 1 antisense RNA 1 (MZF1-AS1) as a novel lncRNA associated with the progression of NB. To investigate the mechanisms underlying the oncogenic functions of MZF1-AS1, we employed the Illumina HiSeq X Ten as a discovery platform to analyze the transcriptome profiling changes of human SH-SY5Y cells in response to stable over-expression of MZF1-AS1. The results showed that stable over-expression of MZF1-AS1 led to altered expression of 2920 human mRNAs, including 1476 up-regulated genes and 1444 down-regulated genes. Then we found the possible roles of these differentially regulated mRNAs in selected pathways including proline synthesis, invasion, and metastasis by Bioinformatic analysis. Furthermore, we validated the RNA-seq results by real-time RT-PCR with high identity. Overall, our results provided fundamental information about the transcriptomic changes in response to MZF1-AS1 over-expression in human NB cells, and these findings will help us understand the pathogenesis of NB. Overall design: Total RNA of cells stably transfected with empty vector or MZF1-AS1 was extracted using the TRIzol® reagent according to the manufacturer's instructions. RNA concentration was measured using a Qubit® RNA Assay Kit with a Qubit® 2.0 Fluorometer (Life Technologies, Inc.), and integrity was assessed using the RNA Nano 6000 Assay Kit with a Bioanalyzer 2100 system (Agilent Technologies, CA). Library preparation and transcriptome sequencing on an Illumina HiSeq X Ten platform were performed by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China), and 100 bp paired-end reads were generated. HTSeq v0.6.0 was used to count the reads numbers mapped to each gene.

神经母细胞瘤（Neuroblastoma, NB）是一类起源于原始神经嵴细胞的恶性胚胎性肿瘤，在儿童恶性肿瘤中占比超过7%，约占儿童癌症相关死亡病例的15%。深入阐明其肿瘤发生与侵袭性的分子机制，对提升神经母细胞瘤的临床治疗效率具有重要意义。本研究通过挖掘公共数据集，鉴定出髓系锌指蛋白1反义RNA1（myeloid zinc finger 1 antisense RNA 1, MZF1-AS1）作为一种与神经母细胞瘤进展相关的新型长链非编码RNA（long non-coding RNA, lncRNA）。为探究MZF1-AS1发挥致癌功能的潜在分子机制，我们以Illumina HiSeq X Ten为测序发现平台，分析了稳定过表达MZF1-AS1的人SH-SY5Y细胞的转录组谱变化。结果显示，MZF1-AS1的稳定过表达可导致2920个人类mRNA的表达发生改变，其中上调基因1476个，下调基因1444个。随后通过生物信息学分析，我们明确了这些差异表达mRNA在脯氨酸合成、侵袭与转移等选定通路中的潜在作用。此外，我们采用实时逆转录聚合酶链反应（real-time RT-PCR）对RNA测序结果进行了验证，二者一致性极高。综上，本研究提供了人神经母细胞瘤细胞中MZF1-AS1过表达所引发的转录组变化的基础数据，上述发现将有助于我们深入理解神经母细胞瘤的发病机制。整体实验设计：按照试剂盒说明书，使用TRIzol®试剂提取稳定转染空载体或MZF1-AS1的细胞总RNA。采用Qubit® RNA检测试剂盒结合Qubit® 2.0荧光分光光度计（Life Technologies公司）测定RNA浓度，使用RNA Nano 6000检测试剂盒结合Agilent 2100生物分析仪（美国加利福尼亚州安捷伦科技公司）评估RNA完整性。文库构建及Illumina HiSeq X Ten平台转录组测序由北京诺禾致源生物信息科技有限公司（中国北京）完成，最终获得100 bp双端测序读段。使用HTSeq v0.6.0软件统计比对至每个基因的读段数目。