Page 162 - Molecular features of low-grade developmental brain tumours

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CHAPTER 6
Cell-cycle analysis
At 24 hours after transfection, cells were collected and fixed in 90% ethanol at 4°C for another 24 hours. Fixed cells were then washed twice with PBS, resuspended in 200 μl PBS containing 1 mg/ml propidium iodide and 1 g/ml RNase and incubated for 10 min at 37°C. Cell cycle analysis was performed using the Fluorescent-Activated Cell Sorter Canto II (BD FACSCanto II, BD Biosciences, San Jose, CA, USA).
Statistical analysis
Statistical analyses were performed with Graphpad Prism® software (Graphpad software Inc., La Jolla, CA, USA). Continuous variables were described with mean and ranges; categorical variables with proportions and percentages. The non-parametric Mann-Whitney test was used to assess differences between two groups. For multiple groups, the non-parametric Kruskal–Wallis test was used followed by a Mann-Whitney test to assess differences between groups. The spearman’s rank correlation test was used to assess the correlation between miR-519d-3p and miR-4758-3p. Group differences and correlations were considered significant if p<0.05.
Results
Mutation analysis of GG and DNT
The BRAFV600E mutation was found in 16 of the 26 (61.5%) GG samples. An initial screen using sanger sequencing identified 11 GG samples that were positive for the BRAFV600E mutation. Samples that were deemed BRAFV600E negative based on sanger sequencing and of which a sufficient amount of DNA was still available were screened further using a next- generation sequencing panel. Based on this panel an additional 5 GG samples were found to be positive for BRAFV600E. One BRAFV600E positive GG also had an additional BRAF mutation (BRAF T559R) on the same allele. A total of 8 DNTs were also screened on the panel of which 1 was found positive for the BRAFV600E, 1 was found positive for FGFR1 mutations (FGFR1 G539R and FGFR1 K656E) and 1 was found positive for CIC G935R mutation of which the pathological implication is unknown.
miR-519d and miR-4758 expression in GG
First, we performed a global expression analysis of 1891 miRNAs using an Exiqon miRCURY LNATM microRNA Array on 11 frozen brain samples (control cortex, n=5; GGs, n=6). Compared to control cortex, a total of 5 miRNAs: miR-519d, miR-4758, miR-664b, miR-4714 and miR-5681b were differentially expressed in GGs. We further validated these 5 miRNAs using Taqman miRNA assays in a larger cohort (frozen samples, control cortex, n=7; GG, n=14). Only miR-519d and miR-4758 were confirmed to be differentially expressed in GGs compared to control cortex, showing 5-fold and 13-fold upregulation (p=0.0335 and p=0.0153, respectively; Fig. 1 A-B). Receiver operating characteristic (ROC) analysis for miR- 519d yielded an area under the curve (AUC) of 0.796, p=0.031 and for miR-4758 AUC=0.837, p=0.0139. Furthermore, a positive correlation was observed between the two miRNAs