Serum long noncoding RNA HOTAIR as a novel diagnostic and prognostic biomarker in glioblastoma multiforme

Abstract

Glioblastoma multiforme (GBM) is the most common and aggressive malignant adult primary brain tumor. Despite surgical resection followed by radiotherapy and chemotherapy, the median survival rate is approximately 14 months. Although experimental therapies are in clinical trials for GBM, there is an urgent need for a peripheral GBM biomarker for measuring treatment response. As we have previously demonstrated that the long noncoding RNA HOX Transcript Antisense Intergenic RNA, or HOTAIR, is dysregulated in GBM and required for GBM cell proliferation, we hypothesized that HOTAIR expression may be utilized as a peripheral biomarker for GBM. HOTAIR expression was measured in serum from 43 GBM and 40 controls using quantitative real-time PCR (qRT-PCR). The PCR products were subsequently subcloned into pCR™4-TOPO®TA vectors for DNA sequencing. A ROC curve was also generated to examine HOTAIR's prognostic value. The amount of HOTAIR in serum exosomes and exosome-depleted supernatant was calculated by qRT-PCR. The relative HOTAIR expression was also investigated in 15 pairs of GBM serum and tumors. We detected HOTAIR in serum from GBM patients. HOTAIR levels in serum samples from GBM patients was significantly higher than in the corresponding controls (P < 0.0001). The area under the ROC curve distinguishing GBM patients from controls was 0.913 (95% CI: 0.845-0.982, P < 0.0001), with 86.1% sensitivity and 87.5% specificity at the cut-off value of 10.8. HOTAIR expression was significantly correlated with high grade brain tumors. In addition, Pearson correlation analysis indicated a medium correlation of serum HOTAIR levels and the corresponding tumor HOTAIR levels (r = 0.734, P < 0.01). We confirmed via sequencing that the amplified HOTAIR from serum contained the HOTAIR sequence and maps to the known HOTAIR locus at 12q13. The serum-derived exosomes contain HOTAIR and the purified exosomes were validated by western blot and nanoparticle tracking analysis. Importantly, our results demonstrate that serum HOTAIR can be used as a novel prognostic and diagnostic biomarker for GBM.

Keywords: Biomarker; Cancer; Glioblastoma; HOTAIR; Long noncoding RNA.

Fig. 1

HOTAIR is detected in serum of GBM patients. a HOTAIR levels are higher in serum from brain tumor patients relative to controls. Serum from normal patients (n = 40) or patients suffering from either pilocytic astrocytoma (n = 6), diffuse astrocytoma (n = 6), GBM (n = 43), oligodendroglioma (n = 4), anaplastic oligodendroglioma (n = 7), were analyzed for the levels of HOTAIR. HOTAIR levels were detected by qRT-PCR and normalized to the corresponding GAPDH levels and relative to an internal control serum. b HOTAIR expression in GBM serum is significantly higher than in normal control patients. The expression level of serum HOTAIR in 43 GBM and 40 control patients were detected and analyzed using Mann-Whitney test (P < 0.001). c Serum HOTAIR can be a significant diagnostic indicator for GBM patients. Serum HOTAIR can discriminate GBM and normal patients with an area under the ROC curve value of 0.913 (P < 0.0001). Note that sensitivity and specificity of the assay was 86.1% and 87.5%, respectively. d Serum HOTAIR levels correlate with GBM tumors. The HOTAIR levels in 15 paired tumors and serum from GBM patients were measured by qRT-PCR. Pearson correlation analysis demonstrated a medium correlation of serum HOTAIR levels and the corresponding tumor HOTAIR levels (r = 0.7342, P < 0.01)

Fig. 2

HOTAIR is enriched in exosome fraction of GBM serum. Exosomes were purified as previously described and the level of HOTAIR was measured by qRT-PCR. a, b Exosomes were found to be 100 nM or smaller (gray regions) in size as judged by NanoSight analysis. c Exosome-enriched protein CD63 was detected by Western blot analysis. d HOTAIR was present in whole serum and purified exosomes but not in serum supernatant depleted of exosomes. e Sequencing of HOTAIR qRT-PCR products amplified from GBM serum demonstrates the HOTAIR sequence. We amplified HOTAIR in GBM patient serum using qRT-PCR, followed by subcloning the corresponding PCR products into pCR™4-TOPO®TA vector and sequencing the inserts. We found that the inserts indeed contained HOTAIR sequence, f that it corresponds to the predicted HOTAIR sequence, and g mapped to the HOTAIR locus on the USC browser