Coding and noncoding landscape of extracellular RNA released by human glioma stem cells

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.

Extracellular RNA profiles with human age

“…Recent analysis of exRNA secreted in extracellular vesicles (EVs) from glioblastoma cells in vitro indicates that ncRNA composes the majority of exRNA species compared with mRNA (Wei et al., 2017). To establish whether this trend is similar for exRNA in serum, we categorized the transcript biotypes into either ncRNA or protein‐coding RNA using ENSEMBL classifications (Supporting Information Table S2).…”

“…In both young and old women, ncRNAs were more abundant than mRNAs (Figure 2). These data are consistent with a recent report comparing RNA isolated from cells and EVs from glioma stem cells (Wei et al., 2017). However, we also observed age‐related differences among snoRNAs.…”

“…A limited number of papers have examined whether circRNAs may be present in serum, mostly using computational methods (Amorim et al., 2017; Gu et al., 2017; Wei et al., 2017). Here, we have used a combination approach and identified 133 extracellular circRNAs (ex‐circRNAs) and further validated age‐related changes in three different ex‐circRNAs: circ_1305 , circ_722, and circ_1445 .…”

“…In general, the most well‐studied biotype of exRNA has been microRNAs (miRNAs), but other noncoding RNAs (ncRNAs) are also present in extracellular fluids including Piwi‐interacting RNAs (piRNAs), long noncoding RNAs (lncRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), Y‐RNAs, and circular RNAs (circRNAs) (Amorim et al., 2017; Wei et al., 2017; Yeri et al., 2017). Although not as well‐characterized, fragments or transcripts of messenger RNA (mRNA) have also been identified in extracellular fluid (Amorim et al., 2017; Wei et al., 2017). …”

“…miRNAs are resistant to nucleases due to their small size and the fact that in body fluids, they are likely protected in extracellular evesicles (EVs) or bound to protein or high‐density lipoprotein (HDL) binding partners (Brase, Wuttig, Kuner & Sultmann, 2010; Cortez et al., 2012; Reid, Kirschner & van Zandwijk, 2011; Wagner et al., 2013). This reservoir of miRNAs may transit to other cells and tissues where in some cases they influence gene regulation and may play a role in physiology and pathology (Hergenreider et al., 2012; Wei et al., 2017; Zhang et al., 2010, 2015). Earlier, we found that several circulating miRNAs are lower in abundance as humans age (Noren Hooten et al., 2010, 2013).…”