Transcriptome profiling using RNA-seq to unravel the aetiology of medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children and accounts for approximately 20% to 25% of all pediatric central nervous system tumors. The current prediction of the prognosis is still highly dependent on clinical characteristics such as the age of the patient, tumor type, stage and localization. Therefore, increased knowledge about the genetic and biological features of these tumors may point to new rationales needed to optimize the diagnosis and treatment of these patients. High expression of the MYC gene promotes cell migration and induces metastatic tumors, which recapitulates the aggressive histological features of cMYC-amplified primary human medulloblastoma. The total extent of transcriptional changes that can be triggered by Myc is remarkable and involves thousands of genes. Although the majority of these effects are not direct, many of the indirect targets are likely to have important roles in mediating the elicited cellular phenotypes. For the induction of the c-Myc protein cultured UW-228 MycER cells were treated with 4-hydroxytamoxifen (500nM) and the transcriptional response was studied in comparison to the vehicle -treated (DMSO) cells after 24, 48 and 72 h. Total RNA was extracted from the cultured cells (triplicate), including start point cells and used for whole transcriptome sequencing (RNAseq) using Illumina HiSeq2500 system. Illumina RNA-Seq from 21 samples generated more than 10 million 100 bp paired-end reads (range 9.728.237- 12.911.421) per library. 73-81.5 % of these reads mapped to the human genome assembly (build 37) and the number of reads mapping to each gene were counted using existing human gene annotations. We performed six pairwise tests of differential gene expression between myc-induced and control cells (3 time points) and between myc-induced cells from different time points. We identified 227 genes, that were significant (Benjamini-Hochberg adjusted p<0.05) in all six pairwise comparisons (time points and treatments). Expression profiling after 72h revealed broad changes in gene expression (90 and 137 up-regulated and down-regulated transcripts, respectively) associated with c-Myc activation. Functional analysis of sets of differentially expressed genes suggested that among the most extensively over-represented pathways were those involved in many different biologic mechanism for example: cell adhesion: chemokines (p=2.138e-20), ECM remodelling (7.477e-14), integrin-mediated cell adhesion and migration (9.943e-13); cytoskeleton remodelling (2.671e-17); development and regulation of epithelial to mesenchymal transition (1.307e-10) and neurophysiological process and receptor-mediated axon growth repulsion (2.095e-10). The most significant outcome of the present work is that it lays out a roadmap for future studies of regulatory mechanisms of medulloblastoma biology by providing a list of key c-Myc candidate target genes for functional analysis.