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Short non-coding RNA sequencing of glioblastoma extracellular vesicles

  • Laboratory Investigation
  • Published:
Journal of Neuro-Oncology Aims and scope Submit manuscript

Abstract

Introduction

Like all nucleated cells, glioblastoma (GBM) cells shed small membrane-encapsulated particles called extracellular vesicles (EVs). EVs can transfer oncogenic components and promote tumor growth by transferring short non-coding RNAs, altering target cell gene expression. Furthermore, GBM-derived EVs can be detected in blood and have potential to serve as liquid biopsies.

Methods

EVs were harvested from culture supernatants from human GBM cell lines, purified via sequential centrifugation, and quantified by nanoparticle tracking. RNA was isolated and short non-coding RNA was sequenced. Data was analyzed via the OASIS-2.0 platform using HG38. MirTarBase and MirDB interrogated validated/predicted miRNA-gene interactions respectively.

Results

Many short non-coding RNA’s were identified within GBM EV’s. In keeping with earlier reports utilizing GBM EV micro-RNA (miRNA) arrays, these included abundant micro-RNA’s including miR-21. However, RNA sequencing revealed a total of 712 non-coding RNA sequences most of which have not been associated with GBM EV’s previously. These included many RNA species (piRNA, snoRNA, snRNA, rRNA and yRNAs) in addition to miRNA’s. miR-21-5p, let-7b-5p, miR-3182, miR-4448, let-7i-5p constituted highest overall expression. Top genes targeted by non-coding RNA’s were highly conserved and specific for cell cycle, PI3K/Akt signaling, p53 and Glioma curated KEGG pathways.

Conclusions

Next generation short non-coding RNA sequencing on GBM EV’s validates findings from earlier studies using miRNA arrays but also demonstrates expression of many additional non-coding RNA sequences and classes previously unassociated with GBM. This may yield important insights into pathophysiology, point to new therapeutic targets, and help develop new biomarkers for disease burden and treatment response.

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References

  1. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996

    Article  CAS  Google Scholar 

  2. Parney IF (2012) Basic concepts in glioma immunology. Adv Exp Med Biol 746:42–52. https://doi.org/10.1007/978-1-4614-3146-6_4

    Article  CAS  PubMed  Google Scholar 

  3. Rodrigues JC, Gonzalez GC, Zhang L, Ibrahim G, Kelly JJ, Gustafson MP, Lin Y, Dietz AB, Forsyth PA, Yong VW, Parney IF (2010) Normal human monocytes exposed to glioma cells acquire myeloid-derived suppressor cell-like properties. Neuro Oncol 12:351–365. https://doi.org/10.1093/neuonc/nop023

    Article  CAS  PubMed  Google Scholar 

  4. Roldan GB, Scott JN, McIntyre JB, Dharmawardene M, de Robles PA, Magliocco AM, Yan ES, Parney IF, Forsyth PA, Cairncross JG, Hamilton MG, Easaw JC (2009) Population-based study of pseudoprogression after chemoradiotherapy in GBM. Can J Neurol Sci 36:617–622

    Article  Google Scholar 

  5. Pitt JM, Kroemer G, Zitvogel L (2016) Extracellular vesicles: masters of intercellular communication and potential clinical interventions. J Clin Invest 126:1139–1143. https://doi.org/10.1172/JCI87316

    Article  PubMed  PubMed Central  Google Scholar 

  6. Kowal J, Tkach M, Thery C (2014) Biogenesis and secretion of exosomes. Curr Opin Cell Biol 29:116–125. https://doi.org/10.1016/j.ceb.2014.05.004

    Article  CAS  PubMed  Google Scholar 

  7. Harshyne LA, Nasca BJ, Kenyon LC, Andrews DW, Hooper DC (2016) Serum exosomes and cytokines promote a T-helper cell type 2 environment in the peripheral blood of glioblastoma patients. Neuro Oncol 18:206–215. https://doi.org/10.1093/neuonc/nov107

    Article  CAS  PubMed  Google Scholar 

  8. van der Vos KE, Abels ER, Zhang X, Lai C, Carrizosa E, Oakley D, Prabhakar S, Mardini O, Crommentuijn MH, Skog J, Krichevsky AM, Stemmer-Rachamimov A, Mempel TR, El-Khoury J, Hickman SE, Breakefield XO (2016) Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain. Neuro Oncol 18:58–69. https://doi.org/10.1093/neuonc/nov244

    Article  CAS  PubMed  Google Scholar 

  9. Akers JC, Ramakrishnan V, Kim R, Phillips S, Kaimal V, Mao Y, Hua W, Yang I, Fu CC, Nolan J, Nakano I, Yang Y, Beaulieu M, Carter BS, Chen CC (2015) miRNA contents of cerebrospinal fluid extracellular vesicles in glioblastoma patients. J Neurooncol 123:205–216. https://doi.org/10.1007/s11060-015-1784-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Manterola L, Guruceaga E, Gallego Perez-Larraya J, Gonzalez-Huarriz M, Jauregui P, Tejada S, Diez-Valle R, Segura V, Sampron N, Barrena C, Ruiz I, Agirre A, Ayuso A, Rodriguez J, Gonzalez A, Xipell E, Matheu A, Lopez de Munain A, Tunon T, Zazpe I, Garcia-Foncillas J, Paris S, Delattre JY, Alonso MM (2014) A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool. Neuro Oncol 16:520–527. https://doi.org/10.1093/neuonc/not218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Shao H, Chung J, Lee K, Balaj L, Min C, Carter BS, Hochberg FH, Breakefield XO, Lee H, Weissleder R (2015) Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nat Commun 6:6999. https://doi.org/10.1038/ncomms7999

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wei Z, Batagov AO, Schinelli S, Wang J, Wang Y, El Fatimy R, Rabinovsky R, Balaj L, Chen CC, Hochberg F, Carter B, Breakefield XO, Krichevsky AM (2017) Coding and noncoding landscape of extracellular RNA released by human glioma stem cells. Nat Commun 8:1145. https://doi.org/10.1038/s41467-017-01196-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kumar R, de Mooij T, Peterson TE, Kaptzan T, Johnson AJ, Daniels DJ, Parney IF (2017) Modulating glioma-mediated myeloid-derived suppressor cell development with sulforaphane. PLoS ONE 12:e0179012. https://doi.org/10.1371/journal.pone.0179012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kelly JJ, Stechishin O, Chojnacki A, Lun X, Sun B, Senger DL, Forsyth P, Auer RN, Dunn JF, Cairncross JG, Parney IF, Weiss S (2009) Proliferation of human glioblastoma stem cells occurs independently of exogenous mitogens. Stem Cells 27:1722–1733. https://doi.org/10.1002/stem.98

    Article  CAS  PubMed  Google Scholar 

  15. Thery C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, Antoniou A, Arab T, Archer F, Atkin-Smith GK, Ayre DC, Bach JM, Bachurski D, Baharvand H, Balaj L, Baldacchino S, Bauer NN, Baxter AA, Bebawy M, Beckham C, Bedina Zavec A, Benmoussa A, Berardi AC, Bergese P, Bielska E, Blenkiron C, Bobis-Wozowicz S, Boilard E, Boireau W, Bongiovanni A, Borras FE, Bosch S, Boulanger CM, Breakefield X, Breglio AM, Brennan MA, Brigstock DR, Brisson A, Broekman ML, Bromberg JF, Bryl-Gorecka P, Buch S, Buck AH, Burger D, Busatto S, Buschmann D, Bussolati B, Buzas EI, Byrd JB, Camussi G, Carter DR, Caruso S, Chamley LW, Chang YT, Chen C, Chen S, Cheng L, Chin AR, Clayton A, Clerici SP, Cocks A, Cocucci E, Coffey RJ, Cordeiro-da-Silva A, Couch Y, Coumans FA, Coyle B, Crescitelli R, Criado MF, D’Souza-Schorey C, Das S, Datta Chaudhuri A, de Candia P, De Santana EF, De Wever O, Del Portillo HA, Demaret T, Deville S, Devitt A, Dhondt B, Di Vizio D, Dieterich LC, Dolo V, Dominguez Rubio AP, Dominici M, Dourado MR, Driedonks TA, Duarte FV, Duncan HM, Eichenberger RM, Ekstrom K, El Andaloussi S, Elie-Caille C, Erdbrugger U, Falcon-Perez JM, Fatima F, Fish JE, Flores-Bellver M, Forsonits A, Frelet-Barrand A, Fricke F, Fuhrmann G, Gabrielsson S, Gamez-Valero A, Gardiner C, Gartner K, Gaudin R, Gho YS, Giebel B, Gilbert C, Gimona M, Giusti I, Goberdhan DC, Gorgens A, Gorski SM, Greening DW, Gross JC, Gualerzi A, Gupta GN, Gustafson D, Handberg A, Haraszti RA, Harrison P, Hegyesi H, Hendrix A, Hill AF, Hochberg FH, Hoffmann KF, Holder B, Holthofer H, Hosseinkhani B, Hu G, Huang Y, Huber V, Hunt S, Ibrahim AG, Ikezu T, Inal JM, Isin M, Ivanova A, Jackson HK, Jacobsen S, Jay SM, Jayachandran M, Jenster G, Jiang L, Johnson SM, Jones JC, Jong A, Jovanovic-Talisman T, Jung S, Kalluri R, Kano SI, Kaur S, Kawamura Y, Keller ET, Khamari D, Khomyakova E, Khvorova A, Kierulf P, Kim KP, Kislinger T, Klingeborn M, Klinke DJ 2nd, Kornek M, Kosanovic MM, Kovacs AF, Kramer-Albers EM, Krasemann S, Krause M, Kurochkin IV, Kusuma GD, Kuypers S, Laitinen S, Langevin SM, Languino LR, Lannigan J, Lasser C, Laurent LC, Lavieu G, Lazaro-Ibanez E, Le Lay S, Lee MS, Lee YXF, Lemos DS, Lenassi M, Leszczynska A, Li IT, Liao K, Libregts SF, Ligeti E, Lim R, Lim SK, Line A, Linnemannstons K, Llorente A, Lombard CA, Lorenowicz MJ, Lorincz AM, Lotvall J, Lovett J, Lowry MC, Loyer X, Lu Q, Lukomska B, Lunavat TR, Maas SL, Malhi H, Marcilla A, Mariani J, Mariscal J, Martens-Uzunova ES, Martin-Jaular L, Martinez MC, Martins VR, Mathieu M, Mathivanan S, Maugeri M, McGinnis LK, McVey MJ, Meckes DG Jr, Meehan KL, Mertens I, Minciacchi VR, Moller A, Moller Jorgensen M, Morales-Kastresana A, Morhayim J, Mullier F, Muraca M, Musante L, Mussack V, Muth DC, Myburgh KH, Najrana T, Nawaz M, Nazarenko I, Nejsum P, Neri C, Neri T, Nieuwland R, Nimrichter L, Nolan JP, Nolte-’t Hoen EN, Noren Hooten N, O’Driscoll L, O’Grady T, O’Loghlen A, Ochiya T, Olivier M, Ortiz A, Ortiz LA, Osteikoetxea X, Ostergaard O, Ostrowski M, Park J, Pegtel DM, Peinado H, Perut F, Pfaffl MW, Phinney DG, Pieters BC, Pink RC, Pisetsky DS, Pogge von Strandmann E, Polakovicova I, Poon IK, Powell BH, Prada I, Pulliam L, Quesenberry P, Radeghieri A, Raffai RL, Raimondo S, Rak J, Ramirez MI, Raposo G, Rayyan MS, Regev-Rudzki N, Ricklefs FL, Robbins PD, Roberts DD, Rodrigues SC, Rohde E, Rome S, Rouschop KM, Rughetti A, Russell AE, Saa P, Sahoo S, Salas-Huenuleo E, Sanchez C, Saugstad JA, Saul MJ, Schiffelers RM, Schneider R, Schoyen TH, Scott A, Shahaj E, Sharma S, Shatnyeva O, Shekari F, Shelke GV, Shetty AK, Shiba K, Siljander PR, Silva AM, Skowronek A, Snyder OL 2nd, Soares RP, Sodar BW, Soekmadji C, Sotillo J, Stahl PD, Stoorvogel W, Stott SL, Strasser EF, Swift S, Tahara H, Tewari M, Timms K, Tiwari S, Tixeira R, Tkach M, Toh WS, Tomasini R, Torrecilhas AC, Tosar JP, Toxavidis V, Urbanelli L, Vader P, van Balkom BW, van der Grein SG, Van Deun J, van Herwijnen MJ, Van Keuren-Jensen K, van Niel G, van Royen ME, van Wijnen AJ, Vasconcelos MH, Vechetti IJ Jr, Veit TD, Vella LJ, Velot E, Verweij FJ, Vestad B, Vinas JL, Visnovitz T, Vukman KV, Wahlgren J, Watson DC, Wauben MH, Weaver A, Webber JP, Weber V, Wehman AM, Weiss DJ, Welsh JA, Wendt S, Wheelock AM, Wiener Z, Witte L, Wolfram J, Xagorari A, Xander P, Xu J, Yan X, Yanez-Mo M, Yin H, Yuana Y, Zappulli V, Zarubova J, Zekas V, Zhang JY, Zhao Z, Zheng L, Zheutlin AR, Zickler AM, Zimmermann P, Zivkovic AM, Zocco D, Zuba-Surma EK (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7:1535750. https://doi.org/10.1080/20013078.2018.1535750

    Article  PubMed  PubMed Central  Google Scholar 

  16. Zhao K, Liang G, Sun X, Guanle L (2016) Comparative miRNAome analysis revealed different miRNA expression profiles in bovine sera and exosomes. BMC Genomics 17:630. https://doi.org/10.1186/s12864-016-2962-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Capece V, Garcia Vizcaino JC, Vidal R, Rahman RU, Pena Centeno T, Shomroni O, Suberviola I, Fischer A, Bonn S (2015) Oasis: online analysis of small RNA deep sequencing data. Bioinformatics 31:2205–2207. https://doi.org/10.1093/bioinformatics/btv113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sun Z, Evans J, Bhagwate A, Middha S, Bockol M, Yan H, Kocher JP (2014) CAP-miRSeq: a comprehensive analysis pipeline for microRNA sequencing data. BMC Genomics 15:423. https://doi.org/10.1186/1471-2164-15-423

    Article  PubMed  PubMed Central  Google Scholar 

  19. Cumba Garcia LM, Peterson TE, Cepeda MA, Johnson AJ, Parney IF (2019) Isolation and analysis of plasma-derived exosomes in patients with glioma. Front Oncol. https://doi.org/10.3389/fonc.2019.00651

    Article  PubMed  PubMed Central  Google Scholar 

  20. Xu X, Bao Z, Liu Y, Jiang K, Zhi T, Wang D, Fan L, Liu N, Ji J (2018) PBX3/MEK/ERK1/2/LIN28/let-7b positive feedback loop enhances mesenchymal phenotype to promote glioblastoma migration and invasion. J Exp Clin Cancer Res 37:158. https://doi.org/10.1186/s13046-018-0841-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhang Z, Lu J, Guo G, Yang Y, Dong S, Liu Y, Nan Y, Zhong Y, Yu K, Huang Q (2017) IKBKE promotes glioblastoma progression by establishing the regulatory feedback loop of IKBKE/YAP1/miR-Let-7b/i. Tumour Biol 39:1010428317705575. https://doi.org/10.1177/1010428317705575

    Article  CAS  PubMed  Google Scholar 

  22. Haderk F, Schulz R, Iskar M, Cid LL, Worst T, Willmund KV, Schulz A, Warnken U, Seiler J, Benner A, Nessling M, Zenz T, Gobel M, Durig J, Diederichs S, Paggetti J, Moussay E, Stilgenbauer S, Zapatka M, Lichter P, Seiffert M (2017) Tumor-derived exosomes modulate PD-L1 expression in monocytes. Sci Immunol. https://doi.org/10.1126/sciimmunol.aah5509

    Article  PubMed  Google Scholar 

  23. Parsa AT, Waldron JS, Panner A, Crane CA, Parney IF, Barry JJ, Cachola KE, Murray JC, Tihan T, Jensen MC, Mischel PS, Stokoe D, Pieper RO (2007) Loss of tumor suppressor PTEN function increases B7–H1 expression and immunoresistance in glioma. Nat Med 13:84–88. https://doi.org/10.1038/nm1517

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was funded in part by Brains Together for a Cure, the Mayo Clinic Center for Individualized Medicine and the Department of Neurological Surgery, Mayo Clinic, Rochester, MN.

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Authors and Affiliations

  1. Department of Neurological Surgery, Mayo Clinic, 200 First St SW, Rochester, MN, 55901, USA

    Tristan de Mooij, Timothy E. Peterson, Brandon McCutcheon & Ian F. Parney

  2. Department of Biomedical Informatics, Mayo Clinic, Rochester, MN, USA

    Jared Evans

  3. Department of Immunology, Mayo Clinic, Rochester, MN, USA

    Ian F. Parney

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  1. Tristan de Mooij
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  2. Timothy E. Peterson
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Correspondence to Ian F. Parney.

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No animal studies were performed. Patient tumor samples used to generate differentiated glioblastoma cell lines were obtained with written, informed consent under a protocol reviewed and approved by the Mayo Clinic Institutional Review Board (IRB#12-003458).

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11060_2019_3384_MOESM1_ESM.pptx

Supplementary Figure 1. Network analysis of the 10 most expressed miRNAs (average of 69.8% of total miRNA expression (range: 56.8 – 75.3%) and their approximately 4238 targets. Note that the gene target signatures for these miRNA’s were highly similar with a large degree of overlap and relatively few outliers (individual gray dots). Supplementary file1 (PPTX 621 kb)

11060_2019_3384_MOESM2_ESM.pptx

A) Unsupervised non-hierarchical clustering of short non-coding RNA expression clustered the five cell lines into two groups: BT116/BT120 and BT114/BT132/BT165. B) Mean overall survival from diagnosis was markedly reduced for BT116/BT120 compared to BT114/BT132/BT165, though this did not reach statistical significance (in keeping with the small sample size). Supplementary file2 (PPTX 61 kb)

Supplementary file3 (DOCX 83 kb)

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de Mooij, T., Peterson, T.E., Evans, J. et al. Short non-coding RNA sequencing of glioblastoma extracellular vesicles. J Neurooncol 146, 253–263 (2020). https://doi.org/10.1007/s11060-019-03384-9

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