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Full-coverage landscape of extracellular RNAs, coding and non-coding, secreted by human glioma stem cells

ABSTRACT: Communication between glioblastoma brain tumor (GBM) and its microenvironment alters the parameters of tumor growth and host responses, and may be mediated in part by tumor-secreted RNA. The global repertoire of extracellular RNAs (exRNAs) released by GBM, however, has not been investigated. We have developed a protocol enabling quantitative, minimally biased analysis of vesicular and non-vesicular exRNA complexes, including microvesicles (MVs) and exosomes (collectively called extracelluar vesicles; EVs), as well as ribonucleoproteins (RNPs) and applied it to study exRNA in patient-derived glioma stem-like cultures (GSC). Despite the intertumoral heterogeneity, further exacerbated at the exRNA level, the extracellular complexes exhibit distinct RNA composition, with microvesicles most closely reflecting the cellular transcriptome, and exRNPs exhibiting the most discrete repertoire. Up to 90% of exRNA reads represent fragmented rRNA; the remaining content is enriched in small ncRNA species, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in both EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs are more abundant than ORF regions; nevertheless, some full-length transcripts are present. Overall, there is less than one copy of non-rRNA per EV. Our results suggest that massive EV/exRNA uptake would be required to ensure functional impact of transferred information to the normal recipient cells of the brain and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse vesicular and non-vesicular exRNA species useful for biomarker discovery.

OTHER RELATED OMICS DATASETS IN: PRJNA360129

ORGANISM(S): other sequences Mus musculus Homo sapiens

PROVIDER: GSE93143 | GEO | 2018/01/04

SECONDARY ACCESSION(S): PRJNA360129

REPOSITORIES: GEO

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Project description:Ectopic adenosine triphosphate synthase (eATP synthase) was found on the cell surface to generate extracellular ATP in various cancer cells. Recently using proteomics approach, several reports identified ATP synthase in extracellular vesicles (EVs) which are important in cell-cell communications. However, the functions of ATP synthase in EVs are still unclear. Here we increased the expression of eATP synthase on the plasma membrane of lung cancer A549 cells via 0.1% FBS starvation and collected EVs including microvesicles and exosomes from the culture medium using serial centrifugation. Both of them were assuredly isolated and confirmed by transmission electron microscopy, nano tracking analysis, and specific EV markers. We also used western blot to demonstrate the presence of ATP synthase in EVs. Besides, dot blot experiment revealed that the F1 portion faced towards extracellular space using anti-ATP beta subunit antibody. Under starvation, the expression of eATP synthase was enhanced on cell surface and exosomes as well as increased EV secretion. To further investigate the functions, we first elucidated whether eATP synthase on EV membrane produced ATP by ATP luciferase analysis. Our data showed that there was no significant ATP production. Moreover, we performed proteomic analysis using dimethyl labeling and LC-MS/MS to compare the difference between EVs released by A549 control and starvation. We identified a total of 1504 and 869 proteins in microvesicles and exosomes, respectively. Using 1.5- and 0.67-fold change as the thresholds, there were 171 and 46 differential expressed proteins in microvesicles and exosomes, respectively. Seven ATP synthase subunits in microvesicles and five in exosomes were identified. The proteomic data were further analyzed using gene set enrichment analysis (GSEA) within the C2 (curated gene sets) subclasses and setting 0.25 as FDR cutoff to find out biological functions related to eATP synthase. Among these up-regulated terms, metabolism, cell cycle and apoptosis were enriched, which indicating that starvation influences cell behavior. Additionally, the immune response is upregulated strikingly in exosomes within C2 gene set. One of these immune response pathways, MHC-1 antigen presentation, was previously proved to interact with eATP synthase which could be recognized by several immune cells.

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Full-coverage landscape of extracellular RNAs, coding and non-coding, secreted by human glioma stem cells

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