MicroRNA and mRNA Expression Changes in Glioblastoma Cells Cultivated under Conditions of Neurosphere Formation

Abstract

Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. The study of the pathogenesis of GBM, as well as the development of targeted oncolytic drugs, require the use of actual cell models, in particular, the use of 3D cultures or neurospheres (NS). During the formation of NS, the adaptive molecular landscape of the transcriptome, which includes various regulatory RNAs, changes. The aim of this study was to reveal changes in the expression of microRNAs (miRNAs) and their target mRNAs in GBM cells under conditions of NS formation. Neurospheres were obtained from both immortalized U87 MG and patient-derived BR3 GBM cell cultures. Next generation sequencing analysis of small and long RNAs of adherent and NS cultures of GBM cells was carried out. It was found that the formation of NS proceeds with an increase in the level of seven and a decrease in the level of 11 miRNAs common to U87 MG and BR3, as well as an increase in the level of 38 and a decrease in the level of 12 mRNA/lncRNA. Upregulation of miRNAs hsa-miR: -139-5p; -148a-3p; -192-5p; -218-5p; -34a-5p; and -381-3p are accompanied by decreased levels of their target mRNAs: RTN4, FLNA, SH3BP4, DNPEP, ETS2, MICALL1, and GREM1. Downregulation of hsa-miR: -130b-5p, -25-5p, -335-3p and -339-5p occurs with increased levels of mRNA-targets BDKRB2, SPRY4, ERRFI1 and TGM2. The involvement of SPRY4, ERRFI1, and MICALL1 mRNAs in the regulation of EGFR/FGFR signaling highlights the role of hsa-miR: -130b-5p, -25-5p, -335-3p, and -34a-5p not only in the formation of NS, but also in the regulation of malignant growth and invasion of GBM. Our data provide the basis for the development of new approaches to the diagnosis and treatment of GBM.

Keywords: RNA-seq; cancer stem cells; epithelial to mesenchymal transition; glioblastoma; mRNA; miRNA; neurospheres; pro-neural to mesenchymal transition.

Figure 1

(A) Representative images of MN (“a”, adherent) and NS (“n”, neurospheres) glioblastoma cells before cell collection for NGS RNAseq analysis. (B) Euclidean distance trees of glioblastoma cell cultures constructed from gene expression data subjected to variation stabilizing transformation (VST). The distance trees for the small RNAs and long RNAs cDNA-libraries are shown separately. The complete agglomeration method for clustering was used.

Figure 2

Principal component analysis (PCA) of DESeq2 normalized, variance stabilizing transformed (VST) gene expression data. PC1:PC2 graphs for small RNAs (A), and long RNAs (B) NGS-libraries. Cell culture specific PC1:PC2 points are annotated with colored envelopes. The black arrows show the general trends of PC1:PC2 transition from the MN (“a”, adherent) to the NS state (“n”, neurospheres).

Figure 3

(A) Venn diagrams showing intersections of miRNA sets of BR3 and U87 GBM cultures, separately for miRNAs with increased (Up) or decreased (Down) levels of NS compared to the corresponding adherent cultures. (B) Heatmap of 7 commonly upregulated and 12 commonly downregulated miRNAs in NS vs. MN GBM cells.

Figure 4

(A) Venn diagrams showing intersections of gene sets of BR3 and U87 MG GBM cells, separately for genes with increased (Up) and decreased (Down) levels for NS cultures compared to the corresponding MN. (B) Heatmap of 38 commonly upregulated and 12 commonly downregulated genes in NS vs. MN glioblastoma cells.

Figure 5

Box plots of DESeq2 normalised expression values of mRNAs and miRNAs grouped by GBM cell cultures and coloured red for monolayer (adherent) cultures and cyan for corresponding NS. Differentially expressed transcripts meet the criteria: DESeq2 pval < 0.05, Log2FoldChange > 0 for upregulated or log2FoldChange < 0 for downregulated (for both BR3 and U87 adherent/neurosphere pairs in one direction—unidirectionally “up” or “down”). Arrows on the right show direction of RNA expression changes in GBM NS for the horizontal block of plots.

Figure 6

Integrated network analysis of downregulated miRNAs and their activated target mRNAs in GBM NS, as well as the participation of the latter in cellular processes. Yellow ovals represent miRNAs, gray round rectangles—mRNA-targets, white round rectangles—cellular processes and signaling pathways.

Figure 7

Integrated network analysis of the GBM NS upregulated miRNAs and their targeted downregulated mRNAs, as well as the participation of the latter in cellular processes. Yellow ovals represent miRNAs, gray round rectangles—mRNA-targets, white round rectangles—cellular processes and signaling pathways.