Polysome profiling of mRNA translation in neuroblastoma cells in response to asparagine/alanine supplement
Neuroblastoma, the most common extracranial solid malignancy in infants and young children, originates from aberrant neural crest cells and accounts for 10-15% of childhood cancer deaths. However, the underlying mechanisms regulating neuroblastoma progression remain to be determined. To investigate the mechanisms regulating tumorigenesis and aggressiveness, we employed the Illumina HiSeq X Ten as a discovery platform to analyze the polysome profiling of mRNA translation in neuroblastoma cells in response to asparagine/alanine (Asn/Ala) supplement. The results showed that 618 and 566 translating mRNAs were respectively elevated or reduced in SK-N-BE(2) after Asn/Ala treatment. Furthermore, we validated the polysome profiling results by western blot with high identity. Overall, our results provided fundamental information about the mRNA translation changes in response to Asn/Ala treatment in human neuroblastoma cells, and these findings will help us understand the pathogenesis of neuroblastoma progression. Overall design: Total RNA of cells treated with asparagine/alanine (Asn/Ala) supplement or deprivation 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 Wuhan SeqHealth Technology Co., Ltd. (Wuhan, China), and 100 bp paired-end reads were generated. HTSeq v0.6.0 was used to count the reads numbers mapped to each gene.