Transcriptomic analysis of neuroblastoma cells in response to stable over-expression of small peptide encoded by hepatocyte nuclear factor 4 alpha antisense RNA 1 (HNF4A-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. We identified a small 51-amino acid peptide (sPEP1) encoded by hepatocyte nuclear factor 4 alpha antisense RNA 1 (HNF4A-AS1) in NB cells treated by serum deprivation. To investigate the mechanisms underlying the oncogenic functions of sPEP1, 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 sPEP1. The results showed that stable over-expression of sPEP1 led to altered expression of 2543 human mRNAs, including 2457 up-regulated genes and 86 down-regulated genes. Then we found the possible roles of these differentially regulated mRNAs in selected pathways including stemness, senescence, 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 sPEP1 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 sORF1 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.