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

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

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

The comparison of intra- and inter-individual miRNA expression variability in the plasma samples of 16 HIs.

<p>Variabilities are represented as boxplots showing miRNA expression coefficients of variation (CVs) for each individual and a complete dataset.</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

Overview of metabolite profiles variability found in plasma samples of healthy volunteers by principle component analysis score plot.

<p>Triangle – male volunteer, circle – female volunteer, coloured according to volunteers, labels shows sampling number within one volunteer. PC - principal component.</p

The ontology enrichment analysis of metabolites that were identified as gender, age and BMI related or were identified as highly variable (HV, CV>0.5) in the analysed healthy population subset.

<p>Total number of differentially present (DP) metabolites (p<0.05) related on gender, age, BMI, BMI within male individuals, age within male volunteers (:male), BMI within female volunteers and age within female volunteers (:female) are also given. Fisher's exact test was used to calculate significance of ontology enrichment (p-value is reported; ns – not significant). ALL - all metabolites within the ontology group. Number of differentially present metabolites in each category is given in brackets within each category.</p

The most prominent BMI-related changes in gene expression were found for interleukin 7 receptor (IL7R), insulin-like growth factor binding protein 3 (IGFBP3), defensin 3 (DEFA3) and DO beta major histocompatibility complex, class II (HLA-DOB).

<p>CD4⁺ T cells - open squares, NK cells - closed circles. Error bars represent intra-individual standard error.</p

Average concentrations of two soluble sugars, glucose and mannose, are significantly increased in plasma samples of healthy volunteers with higher BMI values.

<p>p_Glucose = 0.0014, p_mannose = 0.0001. Error bars represent standard error within monthly measurements.</p