Page 139 - Molecular features of low-grade developmental brain tumours

DYSREGULATION OF MMP/TIMP IN SEGA: MODULATION BY MIR-320D IN VITRO
compared to control tissue, whereas MMP16, MMP25 and TIMP4 expression did not change (Figure 2j,k,l). RNA expression of MMPs and TIMPs did not correlate with the age at epilepsy onset, age at surgery or size of the tumour (p>0.05 and -0.7 < r > 0.7 for both RT-qPCR and RNA-Seq data). Furthermore, RNA expression of MMPs and TIMPs was not associated with the clinical features such as gender, TSC mutation, epilepsy or pre-operative seizure frequency (Mann-Whitney U test or, for multiple groups, the non-parametric Kruskal-Wallis test; p>0.05 for both RT-qPCR and RNA-Seq data). MMP19 expression based on the RT-qPCR was higher in recurrent/regrown SEGA compared to non-recurrent/regrown SEGA (Supplementary Figure 1; p=0.0485). However, RNA-Seq data did not show differences between recurrent/ regrown SEGA and non-recurrent/regrown SEGA (p=0.96).
MMP2 and MMP14 protein expression increased in SEGA
As MMP2 and MMP14 had the most abundant RNA expression in SEGA compared to controls they were selected for further analysis at the protein level. To study cellular localization and distribution, and to quantify protein expression of MMP2 and MMP14 in SEGA, immunohistochemistry was performed on SEGA and periventricular control tissue slides (Figure 3a-f). In periventricular control tissue both MMP2 and MMP14 were not detected in glial cells in the white matter. In nearby cortex tissue, low immunoreactivity of MMP2 and MMP14 was observed in neurons (Figure 3a and b). In SEGA, moderate to high immunoreactivity of MMP2 and MMP14 was found in giant cells (Figure 3 c and d). The OD of MMP2 and MMP14 was higher in SEGA as compared to periventricular white matter of controls (Figure 3e and f; p<0.05).
miR-320d as a predictive regulator of MMPs is lower expressed in SEGA compared to control
In order to identify miRNAs that target MMP2, MMP11, MMP14, MMP15 and MMP19, differentially expressed miRNAs with a lower expression in SEGA as compared to controls were evaluated with miRWalk2. Based on small RNA-Seq data, we found 49 miRNAs with lower expression in SEGA as compared to controls. Six miRNAs (miR-320d, miR-320b, miR-320c, miR-625-5p, miR-330-5p and miR-3200-3p) were predicted to target all five MMPs (MMP2, MMP11, MMP14, MMP15 and MMP19) with a higher expression in SEGA as compared to controls (Figure 4a). Spearman correlations using the RNA-Seq data were calculated between each miRNA and their predicted MMP target (Table 4). miR-320d showed the strongest negative correlation with all five MMPs (MMP2: R: -0,484, p=0.010; MMP11: R: -0.459, p=0.016; MMP14: R: -0.572, p=0.002; MMP15: R: -0.471, p=0.013; MMP19: R: -0.433, p=0.024) and was therefore selected for further analysis. Using RT-qPCR the lower expression of miR-320d in SEGA as compared to control samples was validated (Figure 4b). In order to study the downregulation of miR- 320d on a cellular level, in situ hybridization was performed (Figure 4c). In control tissue, miR- 320d is abundant in glial cells in periventricular white matter and neuronal cells, whereas in SEGA miR-320d was not detected. The OD of miR-320d was lower in SEGA as compared to controls (Figure 4d; p<0.05).
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