(1) To characterize the extracellular vesicle (EV) secretome of noncoding (nc) RNA biotypes in medulloblastoma (MB) and diffuse infiltrative pontine glioma (DIPG).

(2) To characterize the microRNA targetome in MB and DIPG tumor lines and to posit on their unique pathobiologic function(s) via pathway analysis.

EVs from 3 DIPG and 4 MB human cell lines were harvested after serum-free culture, purified via sequential centrifugation, and analyzed using a NanoSight 300 particle analyzer.  RNA was collected using miRNAeasy kit and sequenced with Illumina HiSeq 2500. Trimmed reads were aligned to the HG38 reference genome. Known and novel miRNA were quantified using miRDeep2 based on miRBase v21 sequences.  Differential expression analysis was performed with edgeR.  Pathway analysis was performed with Ingenuity Pathway Analysis.

EV secretomes from both MB and DIPG demonstrated discrete ncRNA biotypes, including miRNA, lincRNA, snoRNA, snRNA, Mt_tRNA and Y RNA fragments.  Unexpectedly, EVs in both DIPG and MB were enriched for Y RNA and not the more commonly studied miRNA biotype.  Hierarchical cluster analysis revealed high discrimination in miRNA expression between DIPG and MB cell lines.  The top 5 differentially expressed miRNAs were miR504-5p, miR193b-3p, miR874-3p, miR1296-5p, miR574-3p.  The top five canonical pathways were Molecular Mechanisms of Cancer, Axonal Guidance Signaling, Ephrin Receptor Signaling, PTEN Signaling, Glioblastoma Signaling. 

Pediatric brain tumors exhibit unique EV secretomes and ncRNA cargos that may provide new insights into the brain tumor microenvironment and identification of novel therapeutic candidates, including Y RNA fragments. To our knowledge this is the first study to comparatively characterize the entire small ncRNA transcriptome in EV and parental samples from MB and DIPG.