Page 109 - Molecular features of low-grade developmental brain tumours

THE CODING AND NON-CODING TRANSCRIPTIONAL LANDSCAPE OF SEGA
to the human reference genome GRCh38. Differential expression testing between SEGA samples and control, identified 140 differentially expressed small RNAs of which 72 were under-expressed and 68 over-expressed (Figure 5a, Supplementary Table 5). Among the differentially expressed small RNAs we identified 1 snRNA, 3 snoRNAs, 4 vtRNAs 94 miRNAs, 15 unannotated gene derived small RNAs and 23 intergenic small RNAs (Figure 5b). The majority of the differentially expressed small RNAs were miRNAs (67.2%) of which 49 were under-expressed in SEGA compared to control and 45 were miRNAs were over-expressed (Figure 5b,c). The unannotated gene derived and intergenic small RNAs, as defined in the materials and methods, formed the second and third largest groups of differentially expressed small RNAs, respectively. Overall 10.7% of the differentially expressed small RNAs were intergenic and 16.4% were gene derived.
Next we analyzed which miRNAs could interact with the 92 enriched pathways from GSEA by assessing each pathway using a Fisher’s exact test, for an over-representation of validated miRNA targets of each differentially expressed miRNA. A total of 81 pathways were found to be enriched for validated targets of 45 of the differentially expressed miRNAs (Fisher’s exact test, adjusted p-value<0.05; Figure 5d). miRNA-20a-5p, miRNA-34a-5p, miRNA- 130b-3p and miRNA-181a-5p were selected for validation using RT-qPCR and were found differentially expressed in accordance with the RNA-Seq data (Figure 5e).
In order to identify miRNAs that could target one or more of LAMTOR1, LAMTOR2, LAMTOR3, LAMTOR4 or LAMTOR5, differentially expressed miRNAs were evaluated with miRWalk2. LAMTOR1 was found to be a validated target of miRNA-20a-5p and let-7c-5p, LAMTOR3 was found to be a validated target of miRNA-362-3p and miRNA-548ba and LAMTOR5 was found to be a validated target of miRNA-221-3p and miRNA-501-5p. Spearman correlations using the RNA-Seq data were calculated between each miRNA and their validated identified target. This analysis identified miRNA-20a-5p (adjusted p-value=0.012, rho=-0.55) and miRNA-221-3p (adjusted p-value=0.015, rho=0.51) as potentially interesting regulatory partners of LAMTOR1 and LAMTOR5 respectfully. Based on these results miRNA- 20a-5p was selected for further analysis. Transfection of primary fetal astrocytes with miRNA- 20a-5p mimic showed that miRNA-20a-5p could downregulate LAMTOR1 (p-value=0.0022), LAMTOR2 (p-value=0.0087) and LAMTOR5 (p-value=0.026) but not LAMTOR3 (p-value=0.4848) and LAMTOR4 (p-value=0.0649; Figure 6).
Discussion
In this study we performed parallel sequencing of the coding and non-coding transcriptome of SEGAs from TSC patients. Among the differentially expressed protein-coding genes we identified an enrichment for genes related to the MAPK pathway. ERK activation in SEGA was confirmed on protein level and was also found in tubers. Both the ERK inhibitor U0126 and rapamycin were able to decrease the proliferation of SEGA cells of one SEGA-derived cell culture in vitro. Additionally, we showed that genes related to the Ragulator complex, a complex activating both the MAPK/ERK and mTORC1 pathway, were over-expressed in SEGA compared to control tissue. Therefore, we provide initial evidence of linkage between these
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