Transcriptomic analysis of neuroblastoma cells in response to stable over-expression of Ets-1 promoter-associated noncoding RNA (pancEts-1)

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 a public microarray dataset and rapid amplification of cDNA ends, we identified v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1) promoter-associated noncoding RNA (pancEts-1) as a novel lncRNA associated with the progression of NB. To investigate the mechanisms underlying the oncogenic functions of pancEts-1, 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 pancEts-1. The results showed that stable over-expression of pancEts-1 led to altered expression of 1912 human mRNAs, including 1035 up-regulated genes and 877 down-regulated genes. Then we found the possible roles of these differentially regulated mRNAs in selected pathways including cell cycle/proliferation, 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 pancEts-1 over-expression in human NB cells, and these findings will help us understand the pathogenesis of NB. Total RNA of cells stably transfected with empty vector or pancEts-1 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.