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.

Extracellular vesicles and intercellular communication within the nervous system

“…The list of potential biomarkers that can provide information on glioma status and response to therapy is expanding and includes changes in the transcriptome profile of EV mRNA (111,112) and small noncoding RNAs in sera (113) as well as mutations in isocitrate dehydrogenase (IDH) mRNA (114) and elevated miR-21 levels in CSF EVs (ref. 115 and for review see ref. 88).…”

Spitting out the demons: Extracellular vesicles in glioblastoma

“…10 In keeping with this idea, Akers et al 45 characterized the presence of miR-21 in EVs prepared from cerebrospinal fluid of GBM patients. However, the in-depth determination of the relative abundance of miRNA in plasma-or cerebrospinal fluidisolated EVs reveals the heterogeneity of such biomarkers.…”

Circulating microRNAs as emerging non-invasive biomarkers for gliomas

“…As far as exosome miRNA expression, miR-21 was found to be overexpressed in serum EVs from patients with GBM IV, and reduced after extensive resection of the tumor (17). Notably, levels of exosome miR-21 in CSF allow to discriminate GBM IV from non-oncologic patients (26) and circulating serum levels probably reflect the antiangiogenic effect of therapy with bevacizumab (21). Therefore, exosome-associated miR-21 expression may serve as a platform for glioma biomarker development.…”