Analysis of Glioblastoma Patients' Plasma Revealed the Presence of MicroRNAs with a Prognostic Impact on Survival and Those of Viral Origin

<div><p>Background</p><p>Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions.</p><p>Experimental Procedures</p><p>MicroRNA profiling of 734 human and human-associated viral miRNAs was performed on blood plasma samples from 16 healthy individuals and 16 patients with GBM, using the nCounter miRNA Expression Assay Kits.</p><p>Results</p><p>We identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients’ survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM.</p><p>Conclusion</p><p>The GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients.</p></div

Schematic presentation of the whole workflow.

<p>1. Material preparation – expanding a GBM cell line, laminin grown and enriched in stem-like cells, 2. Llama immunization with whole GBM cells, 3. Construction of a nanobody library, 4. Phage display cycle – enrichment of antigen specific nanobodies on various biological samples (whole protein extract from GBM tissues, membrane protein-enriched fractions from GBM tissues and GBM stem-like cell lines), 5. ELISA - selection of GBM specific nanobodies, 6. Nanobody production – large scale production and purification of specific nanobodies, 7. Nanobody:antigen pairs – immobilizing the nanobodies and binding to the corresponding antigens, 8. Antigen identification by mass spectrometry, 9. Antigen validation by Western blot. See Materials and methods for additional experimental details.</p

Hierarchical clusters of rules for the validated targets of miRNAs detected in the plasma samples of GPs.

<p>Hierarchical clustering of the top 100 statistically significant rules (p≤0.05) is presented. The SegMine rules were derived from genes, representing validated targets of the GBM-related plasma miRNAs. Euclidian distance and Ward’s linkage criteria were used to compute the hierarchy.</p

MiRNAs with altered plasma levels in the GPs group vs. the HIo group.

<p>MiRNAs with altered plasma levels in the GPs group vs. the HIo group.</p

Genes most frequently targeted by miRNAs, correlated to the presence of GBM or patient survival.

<p>The presented genes are validated targets of miRNAs differentially expressed in the plasma samples of GPs and the members of the HIo subgroup and/or correlated to patient survival according to the results of analyses of this study, obtained by using the miRTarBase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125791#pone.0125791.ref032" target="_blank">32</a>]. <i>VEGFA</i>—vascular endothelial growth factor A, <i>HSPA1B</i>—heat shock 70kDa protein 1B, <i>ACTB</i>—actin beta, <i>HSP90AA1</i>—heat shock protein 90kDa alpha (cytosolic), class A member 1, <i>IGF1R</i>—insulin-like growth factor 1 receptor, <i>CCND1</i>—cyclin D1, <i>PTEN</i>—phosphatase and tensin homolog, <i>BCL2</i>—B-cell CLL/lymphoma 2, <i>CCNE1</i>—cyclin E1, <i>CDK4</i>—cyclin-dependent kinase 4, <i>PPIA</i>—peptidylprolyl isomerase A (cyclophilin A), <i>TUBA1B</i>—tubulin, alpha 1b, <i>WEE1</i>—WEE1 G2 checkpoint kinase, <i>CCND2</i>—cyclin D2, <i>CDK2</i>—cyclin-dependent kinase 2, <i>CDK6</i>—cyclin-dependent kinase 6, <i>BIRC5</i>—baculoviral IAP repeat containing 5, <i>EP300</i>—E1A binding protein p300, <i>RRM2</i>—ribonucleotide reductase M2.</p

Relative band intensities of antigens validated with Western blot.

<p>The relative band intentisy of the antigens, TRIM28 and β-actin, was calculated as the ratio between arbitrary units of the band of the antigen and the arbitrary units of the band of the internal control, GAPDH [AU(antigen)/AU(GAPDH)]. Samples: GSCc – cytosolic/nuclear protein fraction isolated from GBM stem-like cell lines; GBMc – cytosolic/nuclear protein fraction isolated from GBM tissues; NBTc – cytosolic/nuclear protein fraction isolated from human brain samples.</p

Log2 expression values of 11 viral miRNAs, differentially expressed in the GP and HIo plasma samples.

<p>The bars represent the mean values and x each analysed plasma sample.</p

Schematic presentation of the structure of TRIM28.

<p>The conserved RBCC motif is present at the N-terminal domain; the variable C-domain includes the PHD and BROMO domains. Image adapted from Hatakeyama <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113688#pone.0113688-Hatakeyama1" target="_blank">[45]</a>. Author's approval was obtained for the use of this image; original image is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113688#pone.0113688.s002" target="_blank">Figure S2</a>.</p

Sequences of the nanobodies that were chosen for the large-scale production.

<p>The amino acid sequence of the CDR3 regions of the nanobodies is given in alphabetic order.</p

Antigen validation.

<p>Western blot membrane after probing with a monoclonal anti-GAPDH antibody (36 kDa band) used as an internal control for amount of protein loaded in each lane, a monoclonal anti-β-actin antibody (48 kDa band) and monoclonal anti-TRIM28 antibody (100 kDa band). Samples: GSCm – membrane protein-enriched extract isolated from GBM stem-like cell lines; GBMm – membrane protein-enriched extract isolated from GBM tissues; NBTm – membrane protein-enriched extract isolated from human brain samples; GSCc – cytosolic/nuclear protein fraction isolated from GBM stem-like cell lines; GBMc – cytosolic/nuclear protein fraction isolated from GBM tissues; NBTc – cytosolic/nuclear protein fraction isolated from human brain samples; GBMw – whole protein extract from GBM tissues; NBTw – whole protein extract from human brain samples.</p