Page 101 - Molecular features of low-grade developmental brain tumours

THE CODING AND NON-CODING TRANSCRIPTIONAL LANDSCAPE OF SEGA
Figure 1. The protein-coding transcriptome of SEGAs. a. A principal component analysis (PCA) of the RNA-Seq data in SEGA (n=19) and periventricular control tissue (n=8) showing that the major source of variability in gene expression is the diagnosis. X-axis: the first principal component (PC); y-axis: the second PC. b. Spearman’s rank correlation matrix of the RNA-Seq data showing separate clustering of SEGAs from control tissue. The scale bar indicates the strength of the correlation with 1 indicating a strong positive correlation (dark blue) and 0 indicating no correlation (dark red) between samples. c. Volcano plot showing the differentially expressed genes (DEGs) (adjusted p-value<0.05) between SEGAs and control tissue. A total of 4621 mRNAs were found to be over-expressed and 4779 under-expressed in SEGA compared to control tissue. d. Spearman’s rank correlation of the fold changes from TSC1 mutated SEGAs compared to the fold changes from TSC2 mutated SEGAs showing a strong correlation (rho=0.89, p-value<0.001). The Venn diagram shows 5292 DEGs in common between TSC1 and TSC2 mutated SEGAs, 721 DEGs were specific for TSC1 mutated SEGAs and 2816 DEGs were specific for TSC2 mutated SEGAs. e. Schematic overview using Cytoscape of pathways enriched in SEGA compared to control tissue. Geometric testing was used to determine if the amount of DEGs was significant (adjusted p-value<0.02) per pathway. Lines indicate genes in common between pathways. f. Graphical representation of over-expressed genes (red) and under-expressed genes (blue) in 25 enriched pathways containing the highest amount of DEGs.
(14-19 gestational weeks) obtained from medically induced abortions. All material was collected from donors from whom written informed consent for the use of the material for research purposes had been obtained by the Bloemenhove clinic. Tissue was obtained in accordance with the Declaration of Helsinki and the Amsterdam UMC (location AMC) Research Code provided by the Medical Ethics Committee of the AMC. The SEGA cell culture was derived from surgical brain specimen obtained from one TSC patient (age at surgery: 25; gender: female; mutation: TSC1) undergoing surgery at Medical University of Vienna (Vienna, Austria). Primary fetal astrocyte-enriched cell cultures and the primary SEGA culture were prepared as previously described 65,66. Briefly, visible blood vessels were removed, after which the tissue was mechanically minced into smaller fragments and enzymatically digested at 37 °C for 30 minutes with 2.5% trypsin (Sigma-Aldrich, St. Louis, MO). Tissue was washed with incubation medium consisting of Dulbecco’s modified Eagle’s medium (DMEM)/ HAM F10 (1:1) medium (Gibco, Life Technologies, Grand Island, NY), supplemented with 50 units/mL penicillin, 50 μg/mL streptomycin, and 10% fetal calf serum (FCS; Gibco, Life Technologies, Grand Island, NY) and passed through a 70-μm mesh filter. The cell suspension was incubated at 37°C, 5% CO2 for 48 hours in order to let glial cells adhere to the culture flask and was then washed with PBS to removeS excess of myelin and cell debris. Cultures were subsequently refreshed twice a week with incubation medium.
Transfection and treatment of cell cultures
SEGA cells were plated and stimulated with a final concentration of 5 μM U0126 (Sigma- Aldrich, Munich, Germany), 0.1 μM rapamycin (LC Laboratories, Woburn, MA, USA) in DMSO (0.05% final DMSO concentration) or vehicle (0.05% DMSO) for 24 hours. Primary astrocyte cells were transfected with mimic pre-miRNA for miRNA-20a-5p (mirVana miRNA mimics, Applied Biosystems, Carlsbad, CA) using Lipofectamine® 2000 transfection reagent (Life Technologies, Grand Island, NY, USA) in a final concentration of 50 nM for 24 hours. Cells treated with lipofectamine without mimic were used as a control.
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