A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool

Abstract

Background: Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and its prognosis remains dismal despite intensive research and therapeutic advances. Diagnostic biomarkers would be clinically meaningful to allow for early detection of the tumor and for those cases in which surgery is contraindicated or biopsy results are inconclusive. Recent findings show that GBM cells release microvesicles that contain a select subset of cellular proteins and RNA. The aim of this hypothesis-generating study was to assess the diagnostic potential of miRNAs found in microvesicles isolated from the serum of GBM patients.

Methods: To control disease heterogeneity, we used patients with newly diagnosed GBM. In the discovery stage, PCR-based TaqMan Low Density Arrays followed by individual quantitative reverse transcriptase polymerase chain reaction were used to test the differences in the miRNA expression levels of serum microvesicles among 25 GBM patients and healthy controls paired by age and sex. The detected noncoding RNAs were then validated in another 50 GBM patients.

Results: We found that the expression levels of 1 small noncoding RNA (RNU6-1) and 2 microRNAs (miR-320 and miR-574-3p) were significantly associated with a GBM diagnosis. In addition, RNU6-1 was consistently an independent predictor of a GBM diagnosis.

Conclusions: Altogether our results uncovered a small noncoding RNA signature in microvesicles isolated from GBM patient serum that could be used as a fast and reliable differential diagnostic biomarker.

Keywords: GBM; biomarkers; diagnosis; exosomes; sncRNAs.

Fig. 1.

Characterization of MVs isolated from GBM patients’ serum. (A) Transmission electron microscopy analysis. Representative micrograph depicting MVs found in the serum of GBM patients. Arrows point to membrane-like structures with different sizes. (B) Determination of MV size distribution by dynamic light scattering. (C) Evaluation by western blot of the expression levels of several MV/exosome markers in total serum or in MVs isolated from serum of either healthy controls (C) or GBM patients (P). (D) Determination by flow cytometry of CD9 or ECD63 expression on the surface of MVs isolated from healthy controls or GBM patients’ serum. (F) Study design scheme.

Fig. 2.

Expression of sncRNAs in MVs and their association with GBM diagnosis. (A) SncRNA validation by individual quantitative reverse transcription PCR in the training cohort. Expression levels of the individual sncRNAs are normalized to RNU48. Fold change was calculated as the difference of the mean normalized expression values between patients’ and controls’ MVs (−ΔCt = mean (ΔCt GBM patients) – –mean (ΔCt controls)). A t-test analysis was performed with R/Bioconductor. (B) ROC curve showing the true positive and false positive rates for the training cohort for the 3 sncRNA signatures or for each individual one. Validation of the diagnostic value of the sncRNA signature in an independent cohort. AUC, area under the curve. (C) SncRNA expression by individual quantitative reverse transcription PCR in an independent cohort. Expression levels of the individual sncRNAs are normalized to RNU48. Fold change was calculated as the difference of the mean normalized expression values between patients’ and controls’ MVs (ΔCt = mean (ΔCt GBM patients) – –mean (ΔCt controls)). A t-test analysis was performed with R/Bioconductor. (D) ROC curve showing the true positive and false positive rates for the training cohort for the 3 sncRNA signatures or for each individual one.