Exosomal levels of miRNA-21 from cerebrospinal fluids associated with poor prognosis and tumor recurrence of glioma patients

Abstract

Glioma is a most common type of primary brain tumors. Extracellular vesicles, in the form of exosomes, are known to mediate cell-cell communication by transporting cell-derived proteins and nucleic acids, including various microRNAs (miRNAs). Here we examined the cerebrospinal fluid (CSF) from patients with recurrent glioma for the levels of cancer-related miRNAs, and evaluated the values for prognosis by comparing the measures of CSF-, serum-, and exosome-contained miR-21 levels. Samples from seventy glioma patients following surgery were compared with those from brain trauma patients as a non-tumor control group. Exosomal miR-21 levels in the CSF of glioma patients were found significantly higher than in the controls; whereas no difference was detected in serum-derived exosomal miR-21 expression. The CSF-derived exosomal miR-21 levels correlated with tumor spinal/ventricle metastasis and the recurrence with anatomical site preference. From additional 198 glioma tissue samples, we verified that miR-21 levels associated with tumor grade of diagnosis and negatively correlated with the median values of patient overall survival time. We further used a lentiviral inhibitor to suppress miR-21 expression in U251 cells. The results showed that the levels of miR-21 target genes of PTEN, RECK and PDCD4 were up-regulated at protein levels. Therefore, we concluded that the exosomal miR-21 levels could be demonstrated as a promising indicator for glioma diagnosis and prognosis, particularly with values to predict tumor recurrence or metastasis.

Keywords: cancer prognosis; cerebrospinal fluid; extracellular vesicles; glioma; hsa-miR-21.

Figure 1. Characterization of the extracellular vesicles

A. Transmission electronic microscopy of the preparation from patients’ serum and cerebrospinal fluids. Scale bars, 100 nm. B. Western blotting detection for exosomal marker CD63. C. The spectrum from an Agilent Bioanalyzer using RNA extracted from both serum exosomes and CSF exosomes

Figure 2. Comparison of the exosomal miR-21 levels in CSF (n = 70) or serum (n = 50) samples from low- and high-grade glioma patients with the non-glioma controls

Meanwhile, A. Relative CSF miR-21 levels to GAPDH in low-grade (n = 35) and high-grade glioma groups (n = 35) versus in non-glioma patients (n = 25). B. Serum exosomal miR-21 in glioma patients (n = 50) versus non-glioma patients (n = 25). C. Comparison of CSF exosomal miR-21 level and D. Serum exosomal miR-21 between pre-surgery and post-surgery in two patients underwent the second operation due to tumor recurrence. *p < 0.05 and **p < 0.01.

Figure 3. Diagnostic and prognostic values of exosomal miR-21 levels in glioma patients

A. Receiver operation Characterization (ROC) curves based on exosomal miR-21 levels to differentiate recurrent glioma patients (n = 70) from non-tumor diseases. The area under curve (AUC) was as 0.927 (95% CI: 0.865–0.985). The ROC curves to discriminate low grade (Grade II) glioma and high grade (Grade IV or Grade III) cases were shown in B and C. with the AUC of 0.872 (95% CI: 0.817–0.927) and 0.751 (95% CI: 0.681–0.821), respectively. D and E. Kaplan-Meier survival statistics using microarray detection of tissue miR-21 abundance for glioma prognosis (n = 198, from CGGA).

Figure 4. Effects of miR-21 depletion in U251 glioma cells

A. Western blotting of miR-21 target genes of RECK, PDCD4 and PTEN following miR-21 inhibition at 48 h. B. Cellular and exosomal mRNA levels of PTEN, RECK and PDCD4 as determined by RT-qPCR (*p < 0.05, **P < 0.01). C. Flow cytometry of Annexin V staining for the evaluation of cell apoptosis following miR-21 inhibition.