Effect of fibulin-3 knockdown in glioblastoma stem cells

Whole transcriptome comparison of two mesenchymal GBM stem cell lines transfected with control vs fibulin-3 (EFEMP1) siRNAs. GBM stem cell lines GBM09 and GBM34 developed in-house from freshly-resected primary GBM tumors, using standard procecures (Galli et al.; Cancer Res (2002) 64:7011). Cells were transfected with two fibulin-3 or control siRNAs and collected 48h after transfection. Gene expression was quantified by RNAseq using standard procedures. The results suggest that downregulation of fibulin-3 affects NF-kB-dependent GBM gene signatures associated with angiogenesis and immunosuppression. Three independent cultures for each cell line were transfected with two previously validated fibulin-3 siRNAs or control siRNA (Hu et al., Cancer Res (2012) 72:3873). Total RNA was extracted using column purification and poly-A mRNA was enriched and sequenced using Illumina HiSeq 2000. Analysis of gene expression was used to determine a gene signature correlated with fibulin-3 downregulation. Design: 2 GBM stem cell lines x 2 conditions per cell line (control or fibulin-3 KD) x 3 independent replicates. Fibulin-3 mRNA knockdown was confirmed by qRT-PCR of the purified mRNA before RNA sequencing.