Assessment of Small RNA Sorting Into Different Extracellular Fractions
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Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines Juan Pablo Tosar, Fabiana Gámbaro, Julia Sanguinetti, Braulio Bonilla, Kenneth Witwer, Alfonso Cayota To cite this version: Juan Pablo Tosar, Fabiana Gámbaro, Julia Sanguinetti, Braulio Bonilla, Kenneth Witwer, et al.. Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines. Nucleic Acids Research, Oxford University Press, 2015, 43 (11), pp.5601 - 5616. 10.1093/nar/gkv432. pasteur-01881321 HAL Id: pasteur-01881321 https://hal-riip.archives-ouvertes.fr/pasteur-01881321 Submitted on 25 Sep 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International License Published online 04 May 2015 Nucleic Acids Research, 2015, Vol. 43, No. 11 5601–5616 doi: 10.1093/nar/gkv432 Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines Juan Pablo Tosar1,2, Fabiana Gambaro´ 1, Julia Sanguinetti1, Braulio Bonilla1, Kenneth W. Witwer3 and Alfonso Cayota1,4,* Downloaded from https://academic.oup.com/nar/article-abstract/43/11/5601/1172894 by Institut Pasteur user on 01 October 2018 1Functional Genomics Unit, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay, 2Nuclear Research Center, Faculty of Science, Universidad de la Republica,´ Montevideo 11400, Uruguay, 3Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA and 4Department of Medicine, Faculty of Medicine, Universidad de la Republica,´ Montevideo 11600, Uruguay Received January 09, 2015; Revised April 16, 2015; Accepted April 21, 2015 ABSTRACT tween cells (1), extracellular small regulatory RNAs (sR- NAs) circulating in body fluids have attracted great atten- Intercellular communication can be mediated by tion both from a physiological point of view and because of extracellular small regulatory RNAs (sRNAs). Cir- their promising use as minimally invasive disease biomark- culating sRNAs are being intensively studied for ers (2). The potential of plasma or serum miRNA profil- their promising use as minimally invasive disease ing for an earlier cancer diagnosis and to predict progno- biomarkers. To date, most attention is centered on sis and response to therapy has been recently reviewed by exosomes and microRNAs as the vectors and the Schwarzenbach et al. (3). secreted species, respectively. However, this field In order to persist in the extracellular environment would benefit from an increased understanding of and participate in cell-to-cell communication, cell-secreted the plethora of sRNAs secreted by different cell types sRNAs must be protected from ubiquitous extracellular in different extracellular fractions. It is still not clear if RNases. To date, circulating sRNAs have been described in a variety of RNase-insensitive protein or lipid complexes, specific sRNAs are selected for secretion, or if sRNA or encapsulated inside different types of EVs (4). The vast secretion is mostly passive. We sequenced the intra- majority (above 90%) of circulating miRNAs is reportedly cellular sRNA content (19–60 nt) of breast epithelial present in stability-conferring ribonucleoprotein complexes cell lines (MCF-7 and MCF-10A) and compared it with (5,6). Different proteins such as the catalytic core of the extracellular fractions enriched in microvesicles, ex- miRNA-induced silencing complex, Argonaute 2 (5,7)or osomes and ribonucleoprotein complexes. Our re- nucleophosmin 1 (8), have been associated with circulating sults are consistent with a non-selective secretion miRNAs in plasma and cell culture media. Small RNAs model for most microRNAs, although a few showed have also been described in plasma in association with secretion patterns consistent with preferential secre- lipoprotein particles (9). tion. On the contrary, 5 tRNA halves and 5 RNA Y4- However, EV-associated sRNAs have received special at- derived fragments of 31–33 were greatly and signif- tention, in part because of the perceived potential of EVs to interact with and deliver cargo to specific target cells. EVs icantly enriched in the extracellular space (even in include microvesicles, exosomes and apoptotic bodies. Al- non-mammary cell lines), where tRNA halves were though the terms exosomes and microvesicles are often con- ∼ detected as part of 45 kDa ribonucleoprotein com- founded in the literature (10), these EVs differ both in their plexes. Overall, we show that different sRNA families physicochemical parameters (size, density) and in their bio- have characteristic secretion patterns and open the genesis. Exosomes are typically described as having diame- question of the role of these sRNAs in the extracel- ters of 40–100 nm and as being released into the extracel- lular space. lular space by fusion of multivesicular bodies (late endo- somes containing intraluminal vesicles) with the cell mem- INTRODUCTION brane (11). Microvesicles are considered to be larger in size, more irregular in shape and derived from outward blebbing Since the discovery that extracellular vesicles (EVs) can act of the plasma membrane. Finally, apoptotic bodies are de- as vehicles for the exchange of microRNAs (miRNAs) be- *To whom correspondence should be addressed. Tel: +598 25220910; Fax: +598 25224185; Email: [email protected] C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 5602 Nucleic Acids Research, 2015, Vol. 43, No. 11 rived from cells that have undergone programmed cell death ferential and characteristic secretion patterns, raising ques- and contain cellular components such as partially degraded tions about the biological meaning of these non-canonical nucleic acids, peptides and lipids (12). small RNAs in the extracellular space. The cellular mechanisms underlying secretion of sR- NAs and their selection and sorting into different secretory routes are still not fully understood. It has been reported MATERIALS AND METHODS that miRNA secretion is an active ATP-dependent process Cell cultures (8), and that the exporting cell selects specific miRNAs for export (8,13). Other authors have proposed that extracel- Cells were routinely incubated in a humidified chamber at ◦ Downloaded from https://academic.oup.com/nar/article-abstract/43/11/5601/1172894 by Institut Pasteur user on 01 October 2018 lular miRNA levels mirror their intracellular abundances, 37 C with 5% CO2 without antibiotics. MCF-7, MCF-10A and that secretion occurs mostly in a non-selective manner. and NCI-H1299 cells were purchased from ATCC and used < Extracellular sRNAs might even be nothing more than a at low passage ( 10). MCF-7 cells were cultured in EMEM / consequence of necrotic cell death, releasing highly stable (ATCC) + 0.01 mg ml recombinant human insulin + 10% Argonaute/miRNA complexes (7). The different hypothe- FBS (Gibco) until the desired cell number was obtained. ses regarding selective or non-selective miRNA export from After media removal and rinsing with phosphate buffered cells and their roles in cell-to-cell communication have been saline (PBS), cells were changed to defined media [modified / reviewed elsewhere (14,15) and remain a matter of debate. from (26)] consisting of EMEM plus 3.8 g ml recombi- / / The advent of high throughput sequencing has revealed nant human insulin, 0.8 ng ml EGF, 5 g ml transferrin, / ␣ / that the sRNA populations found inside a cell go far be- 475 pg ml PGF2 and 20 g ml fibronectin. After an adap- yond the classical small interfering, piwi-associated and tation period of 48 h, the media was completely removed, miRNA families. Non-protein-coding functional RNAs cells were rinsed with PBS and new defined media was such as rRNA, tRNA, snRNA and snoRNA show terminal added. After further incubation for 48 h, conditioned me- and asymmetric processing to yield smaller RNA entities, dia was collected. Cells (70% confluent) were detached us- involved (or predicted to be involved) in specific regulatory ing a trypsin-EDTA solution, counted and lysed. The same pathways (16). In particular, tRNAs are cleaved near the procedure was followed for HeLa and NCI-H1299 cells. anticodon loop to yield tRNA halves, some of which were MCF-10A cells were cultured in MEGM (Lonza) according shown to inhibit cap-dependent translation initiation and to ATCC recommendations, but addition of cholera toxin to activate a cytoprotective stress-response reprogramming was omitted. After the desired cell number was obtained, protein translation (17). Smaller