Page 137 - Molecular features of low-grade developmental brain tumours

DYSREGULATION OF MMP/TIMP IN SEGA: MODULATION BY MIR-320D IN VITRO
Briefly, blood vessels were removed and the tissue was cut into smaller fragments. The tissue was enzymatically digested by incubating at 37°C for 30 min with trypsin. The enzymatic reaction was stopped by adding twice the amount of astrocyte medium (DMEM/F10, 10% FCS, 5% Penicillin/streptomycin and 5% glutamine) and the cell suspension was passed through a 70-μm cell strainer . After washing two times with astrocyte medium, the cells were incubated at 37°C, 5% CO2 for 48 hours after which they were thoroughly washed with PBS. Fresh medium was added twice a week and cultures reached confluency after 2–3 weeks. Secondary astrocyte cultures for experimental manipulation were established by trypsinizing confluent cultures and sub-plating onto poly-L-lysine (PLL; 15 mg/mL, Sigma- Aldrich, St. Louis, MO, USA)-precoated 6- and 12-well plates (Costar; 50,000 cells/well in a 12-well plate for RNA isolation and RT-qPCR).
Transfection of cell cultures
Cells on PLL-coated plates were transfected with mimic pre-miRNA for hsa-miR-320d (50 μM; Applied Biosystems, Carlsbad, CA, USA) for 24 hours. Cells treated with lipofectamine without mimic were used as a control. Mimics were delivered to the cells using Lipofectamine® 2000 transfection reagent (Life Technologies, Grand Island, NY, USA) in a final concentration of 50 nM.
Statistical Analysis
Statistical analyses were performed with GraphPad Prism version 5 (GraphPad Software, La Jolla, CA, USA) and IBM SPSS Statistics 24 (IBM Corp., Armonk, NY, USA) using the non- parametric Mann-Whitney U test or, for multiple groups, the non-parametric Kruskal-Wallis test followed by Mann-Whitney U test. A p-value < 0.05 was assumed to indicate a statistical difference. Correlations were assessed with R or IBM SPSS Statistics 24, using the Spearman’s rank correlation test (p-value < 0.05 and 0.7 < r > -0.7 was considered statistically significant).
Results
MMP and TIMP expression in SEGA
RNA-Seq data previously produced by our laboratory was used to specifically investigate the expression of MMPs and TIMPs in SEGA 26. In total 19 surgical SEGA samples (obtained from 17 TSC patients and 2 patients without any clinical signs of TSC) and 8 area-matched periventricular controls (autopsy specimens) without a history of other neurological diseases were subjected to RNA-Sequencing 26(Table 1). Differential gene expression analysis identified 4621 genes over-expressed and 4779 under-expressed (adjusted p-value <0.05) in SEGA samples compared to control tissue, of which 133 genes belong to the Reactome based ECM organization pathway (Figure 1a). Furthermore, the gene expression of these 133 genes could be used to separate the control and SEGA samples (Figure 1b). Among the 133 genes identified MMP2, MMP11, MMP14, MMP15, MMP16, MMP17, MMP19, MMP25, TIMP1 and TIMP2 were differentially expressed (Figure 1a,b). Though not present in the ECM organization pathway, the other two TIMPs found in the brain, TIMP3 and TIMP4, were also
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