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      miR-21 in the Extracellular Vesicles (EVs) of Cerebrospinal Fluid (CSF): A Platform for Glioblastoma Biomarker Development

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          Abstract

          Glioblastoma cells secrete extra-cellular vesicles (EVs) containing microRNAs (miRNAs). Analysis of these EV miRNAs in the bio-fluids of afflicted patients represents a potential platform for biomarker development. However, the analytic algorithm for quantitative assessment of EV miRNA remains under-developed. Here, we demonstrate that the reference transcripts commonly used for quantitative PCR (including GAPDH, 18S rRNA, and hsa-miR-103) were unreliable for assessing EV miRNA. In this context, we quantitated EV miRNA in absolute terms and normalized this value to the input EV number. Using this method, we examined the abundance of miR-21, a highly over-expressed miRNA in glioblastomas, in EVs. In a panel of glioblastoma cell lines, the cellular levels of miR-21 correlated with EV miR-21 levels (p<0.05), suggesting that glioblastoma cells actively secrete EVs containing miR-21. Consistent with this hypothesis, the CSF EV miR-21 levels of glioblastoma patients (n=13) were, on average, ten-fold higher than levels in EVs isolated from the CSF of non-oncologic patients (n=13, p<0.001). Notably, none of the glioblastoma CSF harbored EV miR-21 level below 0.25 copies per EV in this cohort. Using this cut-off value, we were able to prospectively distinguish CSF derived from glioblastoma and non-oncologic patients in an independent cohort of twenty-nine patients (Sensitivity=87%; Specificity=93%; AUC=0.91, p<0.01). Our results suggest that CSF EV miRNA analysis of miR-21 may serve as a platform for glioblastoma biomarker development.

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          Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine.

          The clinical performance of a laboratory test can be described in terms of diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy refers to the quality of the information provided by the classification device and should be distinguished from the usefulness, or actual practical value, of the information. Receiver-operating characteristic (ROC) plots provide a pure index of accuracy by demonstrating the limits of a test's ability to discriminate between alternative states of health over the complete spectrum of operating conditions. Furthermore, ROC plots occupy a central or unifying position in the process of assessing and using diagnostic tools. Once the plot is generated, a user can readily go on to many other activities such as performing quantitative ROC analysis and comparisons of tests, using likelihood ratio to revise the probability of disease in individual subjects, selecting decision thresholds, using logistic-regression analysis, using discriminant-function analysis, or incorporating the tool into a clinical strategy by using decision analysis.
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            MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells.

            MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate protein expression by targeting the mRNA of protein-coding genes for either cleavage or repression of translation. The roles of miRNAs in lineage determination and proliferation as well as the location of several miRNA genes at sites of translocation breakpoints or deletions has led to the speculation that miRNAs could be important factors in the development or maintenance of the neoplastic state. Here we show that the highly malignant human brain tumor, glioblastoma, strongly over-expresses a specific miRNA, miR-21. Our studies show markedly elevated miR-21 levels in human glioblastoma tumor tissues, early-passage glioblastoma cultures, and in six established glioblastoma cell lines (A172, U87, U373, LN229, LN428, and LN308) compared with nonneoplastic fetal and adult brain tissues and compared with cultured nonneoplastic glial cells. Knockdown of miR-21 in cultured glioblastoma cells triggers activation of caspases and leads to increased apoptotic cell death. Our data suggest that aberrantly expressed miR-21 may contribute to the malignant phenotype by blocking expression of critical apoptosis-related genes.
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              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.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                21 October 2013
                : 8
                : 10
                : e78115
                Affiliations
                [1 ]Center for Theoretical and Applied Neuro-Oncology, University of California, San Diego, California, United States of America
                [2 ]Exosome Diagnostics, New York, New York, United States of America
                [3 ]Dardinger Laboratory for Neurosciences, Department of Neurosurgery, Ohio State University,Columbus, Ohio, United States of America
                [4 ]Department of Neurosciences, University of California, San Diego, California, United States of America
                [5 ]Neurology Service, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, United States of America
                [6 ]Department of Neurosurgery and Hematology & Medical Oncology, School of Medicine and Winship Cancer Institute, Emory University, Atlanta, Georgia, United States of America
                [7 ]Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China
                City of Hope, United States of America
                Author notes

                Competing Interests: We would like to declare that one of the co-author, Johan Skog, is an employee at Exosome Diagnostics. No other authors have any competing interests. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: JCA VR CCC. Performed the experiments: JCA VR. Analyzed the data: SK SP JK EVM IN RK FH BSC XOB JS. Contributed reagents/materials/analysis tools: SK SP JK EVM IN RK FH BSC XOB JS WH YM. Wrote the manuscript: JCA VR CCC.

                [¶]

                These authors also contributed equally to this work.

                Article
                PONE-D-13-18843
                10.1371/journal.pone.0078115
                3804457
                24205116
                59a5317d-c9d0-4c76-b6b9-7316cc26c5f5
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 May 2013
                : 9 September 2013
                Funding
                CCC is supported by the Doris Duke Charitable Foundation http://www.ddcf.org/, Sontag Foundation http://www.sontagfoundation.com/display.aspx?page=home, Burroughs Wellcome Fund http://www.bwfund.org/, Forbeck Foundation http://wgfrf.org/, and Kimmel Foundation http://www.kimmelfoundation.org/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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