title: MiR-21 in the extracellular vesicles (EVs) of cerebrospinal fluid (CSF): a platform for glioblastoma biomarker development.
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.
authors.affiliation: Center for Theoretical and Applied Neuro-Oncology, University of California, San Diego, California, United States of America.
authors.forename: Johnny C
authors.initials: JC
authors.lastname: Akers
authors.forename: Valya
authors.initials: V
authors.lastname: Ramakrishnan
authors.forename: Ryan
authors.initials: R
authors.lastname: Kim
authors.forename: Johan
authors.initials: J
authors.lastname: Skog
authors.forename: Ichiro
authors.initials: I
authors.lastname: Nakano
authors.forename: Sandeep
authors.initials: S
authors.lastname: Pingle
authors.forename: Juliya
authors.initials: J
authors.lastname: Kalinina
authors.forename: Wei
authors.initials: W
authors.lastname: Hua
authors.forename: Santosh
authors.initials: S
authors.lastname: Kesari
authors.forename: Ying
authors.initials: Y
authors.lastname: Mao
authors.forename: Xandra O
authors.initials: XO
authors.lastname: Breakefield
authors.forename: Fred H
authors.initials: FH
authors.lastname: Hochberg
authors.forename: Erwin G
authors.initials: EG
authors.lastname: Van Meir
authors.forename: Bob S
authors.initials: BS
authors.lastname: Carter
authors.forename: Clark C
authors.initials: CC
authors.lastname: Chen
mesh_terms: D014408:Biomarkers, Tumor / Q000134:cerebrospinal fluid*; D045744:Cell Line, Tumor; D002555:Cerebrospinal Fluid / Q000378:metabolism*; D005909:Glioblastoma / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D014408:Biomarkers, Tumor; C544749:MIRN103 microRNA, human; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D012337:RNA, Ribosomal, 18S