Abstract
Tumor-released RNA may mediate intercellular communication and serve as biomarkers. Here we develop a protocol enabling quantitative, minimally biased analysis of extracellular RNAs (exRNAs) associated with microvesicles, exosomes (collectively called EVs), and ribonucleoproteins (RNPs). The exRNA complexes isolated from patient-derived glioma stem-like cultures exhibit distinct compositions, with microvesicles most closely reflecting cellular transcriptome. exRNA is enriched in small ncRNAs, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs enriched; nevertheless, some full-length mRNAs 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 RNA on brain recipient cells and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse exRNAs useful for biomarker discovery and validates its feasibility on cerebrospinal fluid.
Highlights
Tumor-released RNA may mediate intercellular communication and serve as biomarkers
extracellular vesicles (EVs) and extracellular RNAs (exRNAs) isolation protocols can be generally categorized into three major groups: based on ultracentrifugation (UC), precipitation using a b
Revealing the composition of exRNA complexes released by defined cell types remains one of the most fundamental milestones toward understanding the role of exRNA in intercellular communication, as well as discovery of RNA biomarkers for disease
Summary
Tumor-released RNA may mediate intercellular communication and serve as biomarkers. Here we develop a protocol enabling quantitative, minimally biased analysis of extracellular RNAs (exRNAs) associated with microvesicles, exosomes (collectively called EVs), and ribonucleoproteins (RNPs). MVs and exosomes, broadly called extracellular vesicles (EVs), are released and taken up by various cells, thereby transferring their content. This process likely plays a role in cancer development and manipulation of its microenvironment[4]. Analysis of GSC cellular and extracellular RNA, along with the transcriptome of primary human and mouse cells of the brain microenvironment (neurons, astrocytes, endothelial cells, and microglia) enables us to predict the most impactful miRNAs and expand the repertoire of potentially transferred exRNAs far beyond the classes of miRNAs and mRNAs. We demonstrate that MVs, large vesicles of 0.2–0.8 μm, most closely mirror the cellular transcriptome and present a highly promising but yet poorly explored source of liquid biopsy biomarkers
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