DOCSLIB.ORG

Involvement of Extracellular Vesicle Long Noncoding RNA (Linc-VLDLR) in Tumor Cell Responses to Chemotherapy

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Involvement of Extracellular Vesicle Long Noncoding RNA (Linc-VLDLR) in Tumor Cell Responses to Chemotherapy Published OnlineFirst May 29, 2014; DOI: 10.1158/1541-7786.MCR-13-0636 Molecular Cancer Cell Death and Survival Research Involvement of Extracellular Vesicle Long Noncoding RNA (linc-VLDLR) in Tumor Cell Responses to Chemotherapy Kenji Takahashi, Irene K. Yan, Joseph Wood, Hiroaki Haga, and Tushar Patel Abstract Hepatocellular cancer (HCC) is a highly treatment-refractory cancer and is also highly resistant to adverse cellular stress. Although cell behavior can be modulated by noncoding RNAs (ncRNA) within extracellular vesicles (EV), the contributions of long noncoding RNAs (lncRNAs) are largely unknown. To this end, the involvement and functional roles of lncRNAs contained within EVs during chemotherapeutic stress in human HCC were determined. Expression profiling identified a subset of lncRNAs that were enriched in tumor cell– derived vesicles released from two different cell lines. Of these, lincRNA-VLDLR (linc-VLDLR)was significantly upregulated in malignant hepatocytes. Exposure of HCC cells to diverse anticancer agents such as sorafenib, camptothecin, and doxorubicin increased linc-VLDLR expression in cells as well as within EVs released from these cells. Incubation with EVs reduced chemotherapy-induced cell death and also increased linc-VLDLR expression in recipient cells. RNAi-mediated knockdown of linc-VLDLR decreased cell viability and abrogated cell-cycle progression. Moreover, knockdown of VLDLR reduced expression of ABCG2 (ATP- binding cassette, subfamily G member 2), whereas overexpression of this protein reduced the effects of VLDLR knockdown on sorafenib-induced cell death. Here, linc-VLDLR is identified as an EV-enriched lncRNA that contributes to cellular stress responses. Implications: These findings provide new insight into the role of EVs and demonstrate the capacity of lncRNAs to mediate chemotherapeutic stress response in HCC. Mol Cancer Res; 12(10); 1377–87. Ó2014 AACR. Introduction mechanisms or acquiring resistance to cellular stress Hepatocellular cancer (HCC) is one of the most will enable us to develop more effective treatment for prevalent cancers worldwide, with an annual incidence HCC. of around 750,000 new cases (1). This tumor is char- The major focus of attention in genetic regulation of acterized by the alteration of multiple signaling pathways HCC development, progression, and behavior has been that modulate tumor behavior, local spread, and a ten- on protein-coding genes, and more recently microRNAs dency for multifocal tumor development (2). HCC is (miRNA). In contrast, the contribution of long noncod- ing RNA (lncRNA) to hepatocarcinogenesis has only highly resistant to conventional therapies. Tumor pro- fi gression is increased by the ability of HCC cells to resist recently become appreciated. LncRNAs are de ned as noncoding RNAs more than 200 nucleotides in length adverse environmental stress such as hypoxia, radia- – tion, and chemotherapy. Acquired resistance to adverse (5 8). Like miRNA, these lncRNA can affect regulation environmental conditions enhances tumor propagation, of gene expression and have an impact on many different malignant progression, and resistance to therapy, and cellular processes. In contrast to miRNA, however, they is a central issue in both the pathophysiology and the have complex RNA structures and can function through a therapy of HCC (3, 4). Understanding the primary diverse and broad range of mechanisms. Our previous studies suggested that TUC338, a lncRNA that contains an ultra-conserved element, is significantly increased in Department of Internal Medicine, Department of Transplantation, and human cirrhosis and HCC and can promote cell growth Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida. of HCC cells (9). On the other hand, maternally Note: Supplementary data for this article are available at Molecular Cancer expressed gene 3 is strikingly downregulated in HCC Research Online (http://mcr.aacrjournals.org/). relative to expression in nonmalignant hepatocytes and Corresponding Author: Tushar Patel, Mayo Clinic, 4500 San Pablo Road, playsacrucialroleasatumorsuppressor(10).Although Jacksonville, FL 32224. Phone: 904-956-3257; Fax: 904-956-3359; E-mail: these and other lncRNAs such as MALAT-1, HULC,and [email protected] H19 have been implicated in human HCC, the func- doi: 10.1158/1541-7786.MCR-13-0636 tional contribution of these and other lncRNA genes is Ó2014 American Association for Cancer Research. mostly unknown. www.aacrjournals.org 1377 Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst May 29, 2014; DOI: 10.1158/1541-7786.MCR-13-0636 Takahashi et al. We have recently shown that HCC cells can release performed. The medium was first centrifuged at 300 Â g extracellular vesicles (EV) such as exosomes, which are for 10 minutes, then at 2,000 Â g for 20 minutes in 4Cto membrane-derived vesicles that originate from endosomal remove cells and cell debris. The supernatant was then multivesicular bodies and have a size range of 40 to 100 nm centrifuged at 10,000 Â g for 70 minutes at 4C. The when released into the interstitial fluid. These vesicles supernatant was further ultracentrifuged at 100,000 Â g contain protein, lipids, and RNA derived from their donor for 70 minutes at 4C to pellet EV, which were then cell cytoplasm (11) and can be taken up by other cells. washed by resuspending in phosphate-buffered saline EVs are considered to be related to cell–cell communica- (PBS) and ultracentrifuged at 100,000 Â g for 70 minutes tion and can transfer their content to modulate cellular in 4C. The final pellet, composed of an EV preparation activities in recipient cells (12, 13). These vesicles have that contained a homogeneous population of EVs, was been reported to be secreted into the medium from a used for isolation of extracellular RNA (exRNA) or other variety of normal or tumor cells in culture. We previously studies, or resuspended with 50 to 100 mLofPBSand documented that HCC cell–derived EVs contain miRNAs stored at À80C. The protein yield was measured using a that can modulate transformed cell behavior in recipient Bicinchoninic Acid Protein Assay Kit (Thermo Fisher cells (14). Similar to miRNAs and mRNAs, HCC cell– Scientific Inc.). The number of EV were examined by derived EVs could contain and transfer lncRNAs. We NanoSight (NanoSight Ltd.). postulated that this intercellular signaling could mediate resistance to chemotherapeutic stress in HCC cells. RNA extraction and analysis fi Our studies identi ed involvement of the lncRNA linc- Total RNA was extracted from HCC cells using TRIzol VLDLR in modulation of chemotherapeutic responses by (Life Technologies) and extracellular RNA (exRNA) was fi tumor cell EVs. These ndings provide several new isolated using ExoQuick-TC (System Biosciences). HCC insights into mechanisms of resistance to chemotherapy cells (1 Â 106) were plated in 11 mL of EV-depleted medium in HCC, and the contribution of extracellular lncRNA- on collagen-coated 10-cm dishes. After 3 to 4 days, the mediated signaling in tumor cell responses to adverse medium was collected and sequentially centrifuged at environmental. 3,000 Â g for 15 minutes to remove cells and cell debris. The supernatant was transferred to a sterile vessel and Materials and Methods combined with 2 mL ExoQuick-TC. After an overnight precipitation at 4C, exRNA was extracted using SeraMir Cell lines, culture, and reagents fi Nonmalignant human hepatocytes were obtained from Exosome RNA Ampli cation Kit (System Biosciences) Sciencell and cultured as recommended by the supplier. according to the manufacturer's instructions. RNA concen- HCC cell lines HepG2, Hep3B, PLC/PRF-5, and Huh-7 tration was measured using NanoDrop ND-2000 (Nano- were obtained from the American Type Culture Collec- Drop Technologies). tion. HepG2.ST were obtained from HepG2 cells by spontaneous transformation(15).Humancholangiocar- Real-time PCR analysis cinoma cell line MzChA-1 were obtained as described RNA was treated with RNase-free DNase I (Qiagen). (16). All cell lines were authenticated. All of the HCC cells One microgram of RNA was reverse transcribed to cDNA were cultured in DMEM high glucose medium (HyClone using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Laboratories), containing 10% fetal bovine serum (FBS) Inc.), and real-time quantitative RT-PCR (qRT-PCR) was and 1% antibiotic–antimycotic (Life Technologies), at performed using a Mx3000p System (Stratagene) to detect 37 Cwith5%CO2. Mz-ChA-1 cells were cultured in mRNA and ncRNA expression with SYBR green I (SYBR CMRL Medium 1066 (Life Technologies) with 10% FBS, Advantage qPCR Premix; Clontech Laboratories, Inc.). The 0 1% L-glutamine, and 1% antibiotic–antimycotic mix. For following PCR primers were used: linc-VLDLR, forward: 5 - 0 0 all studies with EVs, EV-depleted medium was prepared AGCAGTCACATTCATCGCAC-3 , reverse: 5 -GAGG- 0 by centrifuging cell-culture medium at 100,000 Â g AATAGGTGCGAACTGC-3 , ATP-binding cassette half- 0 overnight to spin down any preexisting EV content. transporter (ABCC1), forward: 5 -GAGAGTTCCAAGG- 0 0 Camptothecin and doxorubicin were obtained from Sig- TGGATGC-3 , reverse: 5 -AGGGCCCAAAGGTCTTG- 0 ma-Aldrich, and sorafenib was obtained from Selleck TAT-3 , ATP-binding cassette (ABC), subfamily G member 0 Chemicals. Compounds were dissolved in 100% DMSO 2(ABCG2), forward: 5 -TTCGGCTTGCAACAACTA- 0 0 0 and diluted with culture media to the desired concentra- TG-3 , reverse: 5 - TCCAGACACACCACGGATAA-3 , 0
Load more

Recommended publications
  • RNA Delivery by Extracellular Vesicles in Mammalian Cells and Its Applications
    REVIEWS RNA delivery by extracellular vesicles in mammalian cells and its applications Killian O’Brien 1, Koen Breyne1, Stefano Ughetto1,2, Louise C. Laurent 3,4 ✉ and Xandra O. Breakefield 1 ✉ Abstract | The term ‘extracellular vesicles’ refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA , which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell–cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle- associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo.
    [Show full text]
  • Results of a Worldwide Survey
    JOURNAL OF EXTRACELLULAR VESICLES 2018, VOL. 7, 1535745 https://doi.org/10.1080/20013078.2018.1535745 RESEARCH ARTICLE Towards mechanisms and standardization in extracellular vesicle and extracellular RNA studies: results of a worldwide survey Carolina Soekmadji a, Andrew F. Hill b, Marca H. Waubenc, Edit I. Buzásd,e, Dolores Di Viziof, Chris Gardinerg, Jan Lötvall h, Susmita Sahooi and Kenneth W. Witwer j aDepartment of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Brisbane, Australia; bDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia; cDepartment of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; dDepartment of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary; eMTA-SE Immuno-Proteogenomics Research Groups, Budapest, Hungary; fDivision of Cancer Biology and Therapeutics, Departments of Surgery, Biomedical Sciences, and Pathology and Laboratory Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA; gResearch Department of Haematology, University College London, London, UK; hKrefting Research Centre, University of Gothenburg, Sweden; iDepartment of Medicine, Cardiology, Icahn School of Medicine at Mount Sinai, New York, USA; jDepartments of Molecular and Comparative Pathobiology and Neurology, The John Hopkins University School of Medicine, Baltimore, USA ABSTRACT KEYWORDS The discovery that extracellular vesicles (EVs) can transfer functional extracellular RNAs (exRNAs) Extracellular Vesicles; between cells opened new avenues into the study of EVs in health and disease. Growing interest in extracellular RNA; Exosomes; microvesicles; ectosomes; EV RNAs and other forms of exRNA has given rise to research programmes including but not limited to oncosomes; extracellular the Extracellular RNA Communication Consortium (ERCC) of the US National Institutes of Health.
    [Show full text]
  • Characterization of Nucleic Acids from Extracellular Vesicle-Enriched
    Bart et al. BMC Genomics (2021) 22:425 https://doi.org/10.1186/s12864-021-07733-9 RESEARCH Open Access Characterization of nucleic acids from extracellular vesicle-enriched human sweat Geneviève Bart1, Daniel Fischer2, Anatoliy Samoylenko1, Artem Zhyvolozhnyi1, Pavlo Stehantsev1, Ilkka Miinalainen1, Mika Kaakinen1, Tuomas Nurmi1, Prateek Singh1,3, Susanna Kosamo1, Lauri Rannaste4, Sirja Viitala2, Jussi Hiltunen4 and Seppo J Vainio1* Abstract Background: The human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles. Results: We used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin.
    [Show full text]
  • Heterogeneity and Interplay of the Extracellular Vesicle Small RNA
    www.nature.com/scientificreports OPEN Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome Received: 29 December 2017 Helena Sork 1, Giulia Corso1, Kaarel Krjutskov2,3,4, Henrik J. Johansson 5, Joel Z. Nordin1,6, Accepted: 15 June 2018 Oscar P. B. Wiklander1,6, Yi Xin Fiona Lee 7,10, Jakub Orzechowski Westholm 8, Janne Lehtiö 5, Published: xx xx xxxx Matthew J. A. Wood6,7, Imre Mäger7,9 & Samir EL Andaloussi1,6,7 Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV efects refect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specifc miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49–96%) corresponded to rRNA-, coding- and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.
    [Show full text]
  • Charting Extracellular Transcriptomes in the Human Biofluid RNA Atlas
    bioRxiv preprint doi: https://doi.org/10.1101/823369; this version posted May 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Charting extracellular transcriptomes in The Human Biofluid RNA Atlas 2 3 Eva Hulstaert (1,2,3), Annelien Morlion (1,2), Francisco Avila Cobos (1,2), Kimberly Verniers 4 (1,2), Justine Nuytens (1,2), Eveline Vanden Eynde (1,2), Nurten Yigit (1,2), Jasper Anckaert 5 (1,2), Anja Geerts (4), Pieter Hindryckx (4), Peggy Jacques (5,6), Guy Brusselle (7), Ken R. Bracke 6 (7), Tania Maes (7), Thomas Malfait (7), Thierry Derveaux (8), Virginie Ninclaus (8), Caroline 7 Van Cauwenbergh (8), Kristien Roelens (9), Ellen Roets (9), Dimitri Hemelsoet (10), Kelly 8 Tilleman (11), Lieve Brochez (2,3), Scott Kuersten (12), Lukas Simon (13), Sebastian Karg (14), 9 Alexandra Kautzky-Willers (15), Michael Leutner (15), Christa Nöhammer (16), Ondrej Slaby 10 (17,18,19), Roméo Willinge Prins (20), Jan Koster (20), Steve Lefever (1,2), Gary P. Schroth 11 (12), Jo Vandesompele (1,2)*, Pieter Mestdagh (1,2)* 12 13 1 Center for Medical Genetics, Department of Biomolecular Medicine, Ghent University, C. Heymanslaan 10, 14 9000, Ghent, Belgium 15 2 Cancer Research Institute Ghent (CRIG), Ghent University, C. Heymanslaan 10, 9000, Ghent, Belgium 16 3 Department of Dermatology, Ghent University Hospital, C. Heymanslaan 10, 9000, Ghent, Belgium 17 4 Department of Gastroenterology, Ghent University Hospital, C.
    [Show full text]
  • Exogenous RNA As a Regulatory Signal During a Plant's Interaction
    plants Commentary Exogenous RNA as a Regulatory Signal during a Plant’s Interaction with the Biotic Environment: An Evolutionary Perspective and Future Applications in Agriculture Sergey Ivashuta *, Alberto Iandolino and Greg Watson Bayer AG, Chesterfield, MO 63017, USA; [email protected] (A.I.); [email protected] (G.W.) * Correspondence: [email protected] Abstract: Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene ex- pression in a responsive organism by exogenous RNA. While exogenous RNA transfer between organisms of different kingdoms of life have been unambiguously identified in nature, our under- standing of the biological significance of this phenomenon remains obscure, particularly within an evolutionary context. During the last decade multiple reports utilizing various mechanisms of natural eRNAi phenomena have been attempted to develop new agricultural traits and products including weed, disease and insect control. Although these attempts yielded mixed results, this concept remains extremely attractive for many agricultural applications. To better utilize eRNAi for practical applications, we would like to emphasize the necessity of understanding the biological significance of this phenomenon within an evolutionary context and learn from nature by developing advanced tools to identify and study new cases of exogeneous RNA transfer and eRNAi. In this Citation: Ivashuta, S.; Iandolino, A.; opinion article we would like to look at the exogeneous RNA transfer from an evolutionary perspec- Watson, G. Exogenous RNA as a tive, propose that new cases of exogeneous RNA transfer still remain to be identified in nature, and Regulatory Signal during a Plant’s address a knowledge gap in understanding the biological function and significance of RNA transfer.
    [Show full text]
  • Comprehensive Profiling of Extracellular RNA in HPV‑Induced Cancers Using an Improved Pipeline for Small RNA‑Seq Analysis
    Washington University School of Medicine Digital Commons@Becker Open Access Publications 2020 Comprehensive profiling of extracellular RNA in HPV‑induced cancers using an improved pipeline for small RNA‑seq analysis Fangjia Tong Arlise Andress Gongyu Tang Ping Liu Xiaowei Wang Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs www.nature.com/scientificreports OPEN Comprehensive profling of extracellular RNA in HPV‑induced cancers using an improved pipeline for small RNA‑seq analysis Fangjia Tong1, Arlise Andress1, Gongyu Tang1, Ping Liu1 & Xiaowei Wang1,2,3* Extracellular RNAs (exRNAs) have attracted great attention due to their essential role in cell‑to‑cell communication as well as their potential as non‑invasive disease biomarkers. However, at present, there is no consensus on the best method to profle exRNA expression, which leads to signifcant variability across studies. To address this issue, we established an experimental pipeline for comprehensive profling of small exRNAs isolated from cell culture. By evaluating six RNA extraction protocols, we developed an improved method for robust recovery of vesicle‑bound exRNAs. With this method, we performed small RNA sequencing of exosomes (EXOs), microvesicles (MVs) and source cells from 14 cancer cell lines. Compared to cells, EXOs and MVs were similarly enriched in tRNAs and rRNAs, but depleted in snoRNAs. By miRNA profling analysis, we identifed a subset of miRNAs, most noticeably miR‑122‑5p, that were signifcantly over‑represented in EXOs and MVs across all 14 cell lines. In addition, we also identifed a subset of EXO miRNAs associated with cancer type or human papillomavirus (HPV) status, suggesting their potential roles in HPV‑induced cancers.
    [Show full text]
  • Extracellular RNA in Systemic Lupus Erythematosus Shanshan Liu1†, Dandan Wang1† and Yuan Liu2*
    Liu et al. ExRNA (2019) 1:33 https://doi.org/10.1186/s41544-019-0032-y ExRNA REVIEW Open Access Extracellular RNA in systemic lupus erythematosus Shanshan Liu1†, Dandan Wang1† and Yuan Liu2* Abstract Since the discovery of extracellular RNA (exRNA), it has been shown that exRNAs play a significant role as a transducer in intercellular communication and biomarkers in various diseases. Systemic lupus erythematosus (SLE) is a kind of autoimmune disease that has protean manifestations. The survival and long-term prognosis of patients with SLE has improved in these 5–10 years, while disease pathogenesis is still not clear. Many researchers found the changes in exRNA profile, and exRNAs are likely participating in the process of SLE. In this review, we summarize the current profile and function of exRNA in SLE. Circulating miRNAs, in particular, have been identified as biomarkers for SLE diagnosis. We also explore the function of lncRNA in SLE and the potential correlation with disease progression and activity. These studies show that exRNAs may take parts in the process of SLE and some of them can be used as diagnostic tool for SLE. Keywords: exRNAs, miRNAs, lncRNAs, Systemic lupus erythematosus Background RNAs [1–13]. Among the various types of RNA, many The role of extracellular RNA in the pathogenesis and exRNAs sequencing are focused on small RNA species therapy in systemic lupus erythematosus has been exten- (< 200 nt), especially miRNA [14, 15]. The range of con- sively addressed. This review will summarize the most centration of miRNA in sequencing of exRNA varied recent contents in this field.
    [Show full text]
  • Performance Assessment of Total RNA Sequencing of Human Biofluids And
    www.nature.com/scientificreports OPEN Performance assessment of total RNA sequencing of human biofuids and extracellular vesicles Celine Everaert1,2,7, Hetty Helsmoortel1,2,7, Anneleen Decock1,2, Eva Hulstaert1,2,3, Ruben Van Paemel 1,2, Kimberly Verniers1,2, Justine Nuytens1,2, Jasper Anckaert1,2, Nele Nijs4, Joeri Tulkens2,5, Bert Dhondt2,5,6, An Hendrix 2,5, Pieter Mestdagh1,2 & Jo Vandesompele 1,2,4* RNA profling has emerged as a powerful tool to investigate the biomarker potential of human biofuids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofuids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically difer among biofuids. In conclusion, we are the frst to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofuids and their extracellular vesicles. All human biofuids contain a multitude of extracellular nucleic acids, harboring a wealth of information about health and disease status. In addition to established non-invasive prenatal testing of fetal nucleic acids in mater- nal plasma1, liquid biopsies have emerged as a novel powerful tool in the battle against cancer2.
    [Show full text]
  • Obstacles and Opportunities in the Functional Analysis of Extracellular Vesicle RNA – an ISEV Position Paper Bogdan Mateescua*, Emma J
    JOURNAL OF EXTRACELLULAR VESICLES, 2017 VOL. 6, 1286095 http://dx.doi.org/10.1080/20013078.2017.1286095 ORIGINAL RESEARCH ARTICLE Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper Bogdan Mateescua*, Emma J. K. Kowal b*, Bas W. M. van Balkomc, Sabine Barteld, Suvendra N. Bhattacharyyae, Edit I. Buzásf, Amy H. Buckg, Paola de Candiah, Franklin W. N. Chowg, Saumya Dasi, Tom A. P. Driedonksj, Lola Fernández-Messinak, Franziska Haderkl,m, Andrew F. Hill n, Jennifer C. Jones o, Kendall R. Van Keuren- Jensenp, Charles P. Laiq, Cecilia Lässerr,s, Italia di Liegrot, Taral R. Lunavatr,s, Magdalena J. Lorenowiczu, Sybren L. N. Maasv,ImreMägerw,x, Maria Mittelbrunny,StefanMommaz, Kamalika Mukherjeee, Muhammed Nawazaa, D. Michiel Pegtelab, Michael W. Pfafflac, Raymond M. Schiffelersad, Hidetoshi Taharaae, Clotilde Théryaf, Juan Pablo Tosarag, Marca H. M. Waubenj, Kenneth W. Witwer ah and Esther N. M. Nolte-‘tHoenj aDepartment of Biology, Swiss Federal Institute of Technology Zurich (ETH Zürich), Zurich, Switzerland; bDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA; cDepartment of Nephrology and Hypertension, UMC Utrecht, Utrecht, the Netherlands; dExperimental Asthma Research, Priority Area Asthma & Allergy, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Borstel, Germany; eDepartment of Science and Technology, CSIR-Indian Institute of Chemical Biology,
    [Show full text]
  • Profiling of Extracellular Small Rnas Highlights a Strong Bias Towards Non-Vesicular Secretion
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.01.406207; this version posted December 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Profiling of extracellular small RNAs highlights a strong bias towards non-vesicular secretion Helena Sork1,2,*, Mariana Conceicao3, Giulia Corso1, Joel Nordin1,4, Yi Xin Fiona Lee3,§, Kaarel Krjutskov5,6,7, Jakub Orzechowski Westholm8, Pieter Vader9,10, Marie Pauwels11, Roosmarijn Vandenbroucke11, Matthew JA Wood3,12, Samir EL Andaloussi1,4,#, Imre Mäger2,3,#,* 1 Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden 2 Institute of Technology, University of Tartu, Tartu, Estonia 3 Department of Paediatrics, University of Oxford, Oxford, UK 4 Evox Therapeutics, King Charles House, Oxford, United Kingdom 5 University of Helsinki and Folkhälsan Institute of Genetics, Helsinki, Finland 6 Competence Centre on Health Technologies, Tartu, Estonia 7 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden 8 Department of Biochemistry and Biophysics, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Stockholm University, Solna, Sweden 9 Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, The Netherlands 10 Department of Experimental Cardiology, University Medical Center Utrecht, The Netherlands 11 VIB-UGent Center for Inflammation Research, UGent-VIB Research Building FSVM, Gent, Belgium
    [Show full text]
  • Total Extracellular Small RNA Profiles from Plasma, Saliva, and Urine Of
    www.nature.com/scientificreports OPEN Total Extracellular Small RNA Profiles from Plasma, Saliva, and Urine of Healthy Subjects Received: 18 October 2016 Ashish Yeri1, Amanda Courtright1, Rebecca Reiman1, Elizabeth Carlson1, Taylor Beecroft1, Accepted: 01 February 2017 Alex Janss1, Ashley Siniard1, Ryan Richholt1, Chris Balak1, Joel Rozowsky2, Robert Kitchen2, Published: 17 March 2017 Elizabeth Hutchins1, Joseph Winarta1, Roger McCoy3, Matthew Anastasi3, Seungchan Kim4, Matthew Huentelman1 & Kendall Van Keuren-Jensen1 Interest in circulating RNAs for monitoring and diagnosing human health has grown significantly. There are few datasets describing baseline expression levels for total cell-free circulating RNA from healthy control subjects. In this study, total extracellular RNA (exRNA) was isolated and sequenced from 183 plasma samples, 204 urine samples and 46 saliva samples from 55 male college athletes ages 18–25 years. Many participants provided more than one sample, allowing us to investigate variability in an individual’s exRNA expression levels over time. Here we provide a systematic analysis of small exRNAs present in each biofluid, as well as an analysis of exogenous RNAs. The small RNA profile of each biofluid is distinct. We find that a large number of RNA fragments in plasma (63%) and urine (54%) have sequences that are assigned to YRNA and tRNA fragments respectively. Surprisingly, while many miRNAs can be detected, there are few miRNAs that are consistently detected in all samples from a single biofluid, and profiles of miRNA are different for each biofluid. Not unexpectedly, saliva samples have high levels of exogenous sequence that can be traced to bacteria. These data significantly contribute to the current number of sequenced exRNA samples from normal healthy individuals.
    [Show full text]