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.
Bibliographical noteFunding Information:
This work was supported by the U19 CA179563 grant and U19 administrative supplement through the NIH Common Fund and the Office of Strategic Coordination/Office of the NIH Director (A.M.K. and X.O.B.), R21 NS098051 (A.M.K.), and CA069246 (X.O.B.) grants. We thank Robert Kitchen, Sai Lakshmi Subramanian, Alain Charest, Steven Gould, Thorsten Mempel, and the RNAseq working group of the Extracellular RNA Communication Consortium for valuable insights on the data analysis. We thank Maria Ericsson for TEM, Hongjun Wang for designing and manufacturing syringe pumps, Pavel Ivanov for sharing ANG antibody, Oleg Butovsky for sharing miR-21 knockout mice, and Erik J. Uhlmann for reviewing the manuscript. We thank members of Kri-chevsky and Breakefield laboratories for helpful discussions.
© 2017 The Author(s).