TY - JOUR
T1 - miR-21 in the Extracellular Vesicles (EVs) of Cerebrospinal Fluid (CSF)
T2 - A Platform for Glioblastoma Biomarker Development
AU - Akers, Johnny C.
AU - Ramakrishnan, Valya
AU - Kim, Ryan
AU - Skog, Johan
AU - Nakano, Ichiro
AU - Pingle, Sandeep
AU - Kalinina, Juliya
AU - Hua, Wei
AU - Kesari, Santosh
AU - Mao, Ying
AU - Breakefield, Xandra O.
AU - Hochberg, Fred H.
AU - Van Meir, Erwin G.
AU - Carter, Bob S.
AU - Chen, Clark C.
PY - 2013/10/21
Y1 - 2013/10/21
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84886938691&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84886938691&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0078115
DO - 10.1371/journal.pone.0078115
M3 - Article
C2 - 24205116
AN - SCOPUS:84886938691
SN - 1932-6203
VL - 8
JO - PLoS One
JF - PLoS One
IS - 10
M1 - e78115
ER -