A 4-miRNA signature to predict survival in glioblastomas

Abstract

Glioblastomas are among the most lethal cancers; however, recent advances in survival have increased the need for better prognostic markers. microRNAs (miRNAs) hold great prognostic potential being deregulated in glioblastomas and highly stable in stored tissue specimens. Moreover, miRNAs control multiple genes representing an additional level of gene regulation possibly more prognostically powerful than a single gene. The aim of the study was to identify a novel miRNA signature with the ability to separate patients into prognostic subgroups. Samples from 40 glioblastoma patients were included retrospectively; patients were comparable on all clinical aspects except overall survival enabling patients to be categorized as short-term or long-term survivors based on median survival. A miRNome screening was employed, and a prognostic profile was developed using leave-one-out cross-validation. We found that expression patterns of miRNAs; particularly the four miRNAs: hsa-miR-107_st, hsa-miR-548x_st, hsa-miR-3125_st and hsa-miR-331-3p_st could determine short- and long-term survival with a predicted accuracy of 78%. Heatmap dendrograms dichotomized glioblastomas into prognostic subgroups with a significant association to survival in univariate (HR 8.50; 95% CI 3.06-23.62; p<0.001) and multivariate analysis (HR 9.84; 95% CI 2.93-33.06; p<0.001). Similar tendency was seen in The Cancer Genome Atlas (TCGA) using a 2-miRNA signature of miR-107 and miR-331 (miR sum score), which were the only miRNAs available in TCGA. In TCGA, patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors and low miR sum score had the shortest survival. Adjusting for age and MGMT status, low miR sum score was associated with a poorer prognosis (HR 0.66; 95% CI 0.45-0.97; p = 0.033). A Kyoto Encyclopedia of Genes and Genomes analysis predicted the identified miRNAs to regulate genes involved in cell cycle regulation and survival. In conclusion, the biology of miRNAs is complex, but the identified 4-miRNA expression pattern could comprise promising biomarkers in glioblastoma stratifying patients into short- and long-term survivors.

Fig 1. Principal component analysis and effect of fixation time.

(A) Principal component analysis (PCA) plot showing differences between patients. The data supports our initial observation that sample 10 was a technical outlier. (B, C) Fixation time did not significantly affect the miRNA array data as miRNA data, obtained from a glioblastoma short-term fixated 1 hour and 48 hours, showed a strong correlation for (B) all probes (r_s = 0.98) and (C) human probes (r_s = 0.97).

Fig 2. Short-(STS) and long-term (LTS) glioblastoma survivors have different microRNA (miRNA) profiles.

(A) Heatmap of the ten most deregulated miRNAs in STS and LTS. STS and LTS are grouped into two overall patterns as shown by the dendrograms. Pattern one (red bar) was characterized by STS whereas pattern two (green bar) mostly characterized LTS (18 LTS and 7 STS). In the heatmap, red represents upregulated miRNAs and green represents downregulated miRNAs. (B) Kaplan Meier plot showing a significant separation in overall survival between the two patterns. (C) Volcano plot illustrating that no miRNAs were significantly deregulated above the two-fold threshold. Blue represent normal fold changes and p-values while red represent permutated values. The four miRNAs included in the signature are indicated with arrows.

Fig 3. In silico gene expression analysis and KEGG pathway analysis.

(A) Low summed scores for miR-107 and miR-331-3p (miR sum score) tended to associate with poorer prognosis in the TCGA data set (n = 247) when dichotomized at the median. (B) Glioblastomas with unmethylated MGMT promoter (u-MGMT) had higher miR sum score than glioblastomas with methylated MGMT promoter (m-MGMT). (C) Stratified into groups based on MGMT methylation status and miR sum score, patients with low miR sum score and u-MGMT had the shortest survival. (D) KEGG pathway analysis based on predicted targets of hsa-miR-107, hsa-331-3p, hsa-548x and hsa-3125 in the Glioma Pathway performed using the DIANA-mirPath tool. Genes regulated by at least two miRNAs are indicated with red, genes regulated by one miRNA with yellow, and genes not regulated by any of the miRNAs with blue. (E) Possible mechanisms by which downregulation of the 4-miRNA signature contribute to shorter survival in patients with glioblastoma.