168
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      MicroRNAs are exported from malignant cells in customized particles

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body.

          Related collections

          Most cited references57

          • Record: found
          • Abstract: found
          • Article: not found

          Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.

          Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling. Copyright © 2012 Elsevier Inc. All rights reserved.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Exosomal microRNA: a diagnostic marker for lung cancer.

            To date, there is no screening test for lung cancer shown to affect overall mortality. MicroRNAs (miRNAs) are a class of small noncoding RNA genes found to be abnormally expressed in several types of cancer, suggesting a role in the pathogenesis of human cancer. We evaluated the circulating levels of tumor exosomes, exosomal small RNA, and specific exosomal miRNAs in patients with and without lung adenocarcinoma, correlating the levels with the American Joint Committee on Cancer (AJCC) disease stage to validate it as an acceptable marker for diagnosis and prognosis in patients with adenocarcinoma of the lung. To date, 27 patients with lung adenocarcinoma AJCC stages I-IV and 9 controls, all aged 21-80 years, were enrolled in the study. Small RNA was detected in the circulating exosomes. The mean exosome concentration was 2.85 mg/mL (95% CI, 1.94-3.76) for the lung adenocarcinoma group versus 0.77 mg/mL (95% CI, 0.68-0.86) for the control group (P < .001). The mean miRNA concentration was 158.6 ng/mL (95% CI, 145.7-171.5) for the lung adenocarcinoma group versus 68.1 ng/mL (95% CI, 57.2-78.9) for the control group (P < .001). Comparisons between peripheral circulation miRNA-derived exosomes and miRNA-derived tumors indicated that the miRNA signatures were not significantly different. The significant difference in total exosome and miRNA levels between lung cancer patients and controls, and the similarity between the circulating exosomal miRNA and the tumor-derived miRNA patterns, suggest that circulating exosomal miRNA might be useful as a screening test for lung adenocarcinoma. No correlation between the exosomal miRNA levels and the stage of disease can be made at this point.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Exosome secretion: molecular mechanisms and roles in immune responses.

              Exosomes are small membrane vesicles, secreted by most cell types from multivesicular endosomes, and thought to play important roles in intercellular communications. Initially described in 1983, as specifically secreted by reticulocytes, exosomes became of interest for immunologists in 1996, when they were proposed to play a role in antigen presentation. More recently, the finding that exosomes carry genetic materials, mRNA and miRNA, has been a major breakthrough in the field, unveiling their capacity to vehicle genetic messages. It is now clear that not only immune cells but probably all cell types are able to secrete exosomes: their range of possible functions expands well beyond immunology to neurobiology, stem cell and tumor biology, and their use in clinical applications as biomarkers or as therapeutic tools is an extensive area of research. Despite intensive efforts to understand their functions, two issues remain to be solved in the future: (i) what are the physiological function(s) of exosomes in vivo and (ii) what are the relative contributions of exosomes and of other secreted membrane vesicles in these proposed functions? Here, we will focus on the current ideas on exosomes and immune responses, but also on their mechanisms of secretion and the use of this knowledge to elucidate the latter issue. © 2011 John Wiley & Sons A/S.
                Bookmark

                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                October 2012
                October 2012
                5 July 2012
                5 July 2012
                : 40
                : 18
                : 9125-9138
                Affiliations
                1Department of Cellular and Molecular Pharmacology, Chicago Medical School, 2Department of Pathology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, 3Division of Biology and Biomedical Sciences, Molecular Cell Biology, Washington University School of Medicine, St. Louis, MO, 4Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, 5Department of Cell Biology and Anatomy, Rosalind Franklin University of Medicine and Science, North Chicago, 6Department of Biology, Lake Forest College, Lake Forest, 7University of California, San Diego School of Medicine and 8Rush Medical College, Rush University, Chicago, IL, USA
                Author notes
                *To whom correspondence should be addressed. Tel: +1 847 578 8341; Fax: +1 847 755 6525; Email: Dominik.Duelli@ 123456Rosalindfranklin.edu

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

                Article
                gks656
                10.1093/nar/gks656
                3467054
                22772984
                5ace39fa-9ff9-4207-b56d-dcb97dd3a3b4
                © The Author(s) 2012. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 28 March 2012
                : 12 June 2012
                : 13 June 2012
                Page count
                Pages: 14
                Categories
                Molecular Biology

                Genetics
                Genetics

                Comments

                Comment on this article