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. 2017 Oct 26;8(1):1145.
doi: 10.1038/s41467-017-01196-x.

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

Zhiyun Wei  1 , Arsen O Batagov  2 , Sergio Schinelli  3 , Jintu Wang  4 , Yang Wang  1 , Rachid El Fatimy  1 , Rosalia Rabinovsky  1 , Leonora Balaj  5 , Clark C Chen  6 , Fred Hochberg  7   8 , Bob Carter  7 , Xandra O Breakefield  5 , Anna M Krichevsky  9
Affiliations

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

Zhiyun Wei et al. Nat Commun. .

"VSports app下载" 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 VSports手机版. 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. .

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"VSports注册入口" Conflict of interest statement

X. O V体育安卓版. B. is a consulting member of the Scientific Advisory Board of Evox Therapeutics, Ltd. and Exocyte Therapeutics, Ltd. The remaining authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Flowchart of the exRNA fractionation and sequencing. a The pipeline of the filtration-based exRNA isolation. Following removal of cells and cellular debris by low speed centrifugation, the supernatants were filtered through a sequence of reduced pore sizes (2.0, 0.8, 0.22, and 0.02 μm) to separate the extracellular fractions, and a final concentrator with the cutoff of 3 kDa was applied to collect the remaining small particles. b The aliquots of conditioned media after 0.8 μm filtration were used for MV and exosome isolation, either by ultracentrifugation (UC) or filtration, and the RNA yield of these fractions compared. The number of remaining vesicles/particles was compared in UC supernatant and filter flow-through. N = 4 aliquots of conditioned media. All bars represent mean ± SEM. c Comparison between the filtration-based exRNA isolation and other common exRNA isolation methods. The stars mark superior characteristics of sequential filtration over other methods. d The optimized pipeline for the broad coverage, minimally biased RNA-sequencing. RNA of 15–65 nt was selected for the small RNA libraries, to reduce the overwhelming levels of tRNAs. NS, not significant; *p < 0.05; **p < 0.01; t-test
Fig. 2
Fig. 2
Quality control of the fraction separation. a Transmission electron microscopy of EVs and RNPs isolated using the sequential filtration protocol. TEMs were replicated three times. b Protein markers, identified by the western blotting, verified the separation of extracellular fractions. Equal protein input of 50 μg per lane was used. Western blots were replicated twice. c RNA yields of extracellular fractions from different GSC cultures
Fig. 3
Fig. 3
rRNA depletion and media correction are warranted for the deep RNAseq analysis. a Comparison of the exRNA libraries prepared with and without rRNA depletion. The percent of rRNA reads is shown, indicating that the majority of exRNA reads in non-depleted libraries represent rRNA. Sequencing depth of other exRNA classes is substantially increased by the rRNA depletion. b Quantification of total RNA in conditioned and fresh media indicates that the vast majority is cell derived (n = 4 GSC cultures). Nevertheless, quantification of specific RNA species can be skewed by the media, as illustrated in c. The levels of miR-122 were assessed in exRNA isolated from the fractions of conditioned and fresh media. miR-122 enrichment in exRNA (n = 4 GSC cultures) was calculated pre- and post-correction to the levels in fresh media, as described in Supplementary Fig. 6. miR-122 was highly enriched in the GSC exRNA, relative to its intracellular level, with up to 1500-fold enrichment in the exosomes before media correction. miR-122 enrichment in GSC exosomes became marginal upon correction. All bars represent mean ± SEM
Fig. 4
Fig. 4
Relative composition of diverse RNA classes in cellular and extracellular compartments (MVs, exosomes, and RNPs) in different GSC cultures. a The top panels exhibit relative RNA composition in long RNA libraries, and the bottom panels depict the composition in small RNA libraries. The data were normalized to the total number of annotated non-rRNA reads. The bars framed in red represent the mean values of four GSC cultures. b RT-PCR analysis (with equal input of total RNA) of selected mRNAs abundant in exRNA, demonstrates the presence of nearly full-length short, but not long messages in the extracellular fractions. Long RT-PCRs were replicated twice. c Long RNA libraries-based analysis of the length distribution of 500 most abundant mRNAs suggests no length preference for shorter parent transcripts in the extracellular fractions. d Analysis of the RNAseq reads mapped to mRNAs indicates that UTR regions were more enriched than CDS regions in the extracellular fractions (n = 4 GSC cultures). mRNA reads were aligned to the coding sequences (CDS), 5′-UTRs, and 3′-UTRs separately, and the alignment rates for each extracellular fraction were normalized to the corresponding cellular fraction. The log-transformed ratios of the alignment rates were compared among the three regions. Error bars represent mean ± SEM. NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; t-test
Fig. 5
Fig. 5
Inequality and heterogeneity of the RNA repertoire of extracellular fractions. a Evenness factors, reflecting the inequality of abundance’ distribution of the indicated RNA classes in various fractions. Higher evenness factors correspond to lower inequality. The classes of tRNA, Y RNA, snRNA, and SRP RNA, but not miRNA, contributed to the increased inequality of exRNA most significantly. Differential evenness factors of cellular and extracellular RNA suggest the selectivity of secretion (n = 4 GSC cultures). b For each RNA category, a sum of squared errors (χ 2 value) was calculated among four GBM cultures, after normalization of each RNA species to the total number of reads in that RNA category. The χ 2 value of each extracellular fraction was compared to the cellular fraction. Fold change of χ 2 values higher than 1 reflects the increased heterogeneity. Heterogeneity either increased or decreased more than two-fold is highlighted in red and blue, respectively. c Venn diagrams depict the number of common species among 100 top abundant mRNAs, in four GBM cultures and their extracellular fractions, supporting the observation of higher heterogeneity of mRNA composition in exRNA than in cellular RNA. Significant differences between the cellular and extracellular fractions are depicted as following: *, p < 0.05; **, p < 0.01; ***, p < 0.001; t-test
Fig. 6
Fig. 6
Y RNA and tRNA fragments are abundant and enriched in exRNA. a, b Specific Y RNA and tRNA species are among the most abundant in small RNA libraries (n = 4 GSC cultures). The reads corresponding to Y1, Y4, and Y5 are highly enriched in extracellular fractions, especially in RNP (a). The reads corresponding to specific tRNAs, such as GluCTC and GlyGCC, are highly enriched in exRNA, while others (e.g. GlnTTG and LeuCAA) are not (b). c Mapping coverage of Y1 reads indicates that the Y1 is precisely processed, and mostly its 5′ fragment is present in exosomes and RNPs, as evidenced by the steep peaks corresponding to the 5′-end 30 nt. These profiles are distinct from the more uniform full-length coverage observed for the cellular RNA, and to a lesser extent MV RNA. Similar analysis for other Y RNAs and tRNAs is presented in Supplementary Fig. 12. d The predicted secondary structure of Y1 RNA, Copyright (1993) National Academy of Sciences, USA, and the position of its cleavage (indicated by the arrow) that produces the 5′ fragment which is highly abundant in exRNA. e Quantification of Y RNA reads in long and small RNA libraries, demonstrates different fragment to full-length ratios in the cell and exRNA fractions (n = 3 GSC cultures; MGG75 was excluded due to very low abundance of the full-length Y RNA). The ratios are increased in extracellular fractions. f qRT-PCR analysis with primers specific to either full-lengths or fragments of several RNA species validates the enrichment of 5′ tRNA and Y RNA fragments in extracellular fractions. For each specific transcript examined, two lines represent GBM8 and 20/3 cells, respectively. g qRT-PCR analysis of selected transcripts confirms the quantitative character of the RNAseq pipeline. The blue dots represent qRT-PCR Cq values, while the red dots represent the results of RNAseq quantification in fmol. Both analyses were performed on the same set of RNA samples. Error bars represent mean ± SEM
Fig. 7
Fig. 7
RNA repertoire of MV most closely reflects cellular RNA composition. a Heat map cluster analysis of RNA classes indicates relative similarity of the composition of MVs and the source cells. The scale bar represents the percentage of non-rRNA annotated reads. b PCA analysis of RNA classes. Different fractions are marked in different colors. Within each fraction, four dots represent four GSC cultures. c, d GBM4 MV represents the cellular mRNA content closely, and much better than exosomes or RNPs, based on the correlation analysis of all mRNA species (c), and cluster analysis of the top abundant mRNAs (d). The scale bar represents mRNA abundance. e, f Extracellular miRNA composition, in general, is less reflective of the cellular miRNome; nevertheless, MV fraction still remains the best simulator, based on the correlation analysis of all miRNA species (e), and cluster analysis of the top abundant miRNAs (f). The scale bar represents the log-transformed miRNA abundance. Similar analyses of other cell cultures can be found in Supplementary Figs. 14–17. g qRT-PCR analysis of exRNA fractions isolated from the CSF of GBM patients, using the same filtration-based procedure, indicates its applicability to clinical samples. The data verify preferential association of selected RNA species with different exRNA fractions in the human biofluid (n = 4 CSF samples). All bars represent mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; t-test
Fig. 8
Fig. 8
Comparative analysis of GSC-secreted miRNAs and cellular miRNome of the normal cells of the brain predicts the most impactful GBM miRNAs in tumor-to-microenvironment communication. a Extracellular fractions of GSC cultures were compared to primary human astrocytes (HA), based on the corresponding RNAseq data sets. The fold-changes in miRNA levels were log transformed and a t-test was applied to examine the significance of difference. MiRNAs with log-fold changes higher than 1.7, which corresponded to 50 times higher levels in the GSC-derived exRNA fractions relative to the recipient cells, and p < 0.05 (t-test), were defined as potentially impactful (colored in red). The horizontal axis of Volcano plots shows the log-fold difference, and the vertical axis shows the statistical significance. Similar analyses of primary human neuroglial and endothelial cells are shown in Supplementary Fig. 18. b A full list of most impactful GSC miRNAs for human and mouse astrocytes, neurons, microglia, or brain endothelial cells. The number of “+” symbols reflects the number of extracellular fractions in which an miRNA meets the indicated criteria as in a. Most of these miRNAs are also upregulated in the GBM tumors compared to non-neoplastic brain tissues in the TCGA microarray data set, as indicated in the three right columns (n = 496 GBM vs 10 control). c Top enriched IPA pathways for the validated mRNA targets of the most impactful miRNAs. Predicted activation and inhibition of pathways are labeled as orange and blue bars, respectively. The yellow line shows the percent of genes in each pathway that are validated targets. d, e Co-cultured with GBM8 neurospheres, primary miR-21-null astrocytes exhibit steady miR-21 levels (d) and downregulation of validated miR-21 targets (e). Cq value of miR-21 in mono-cultures was defined as 45 (undetectable expression). N = 4 wells in 24-well plate. All bars represent mean ± SEM. *, p < 0.05; **, p < 0.01; t-test
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