Scratch Assay Cells were transfected as described with miRNAs miR-34c-5p, miR-185 and miR-190, and plated at a density of 2.4��105 to allow for confluency at 48 hours. Forty-eight hours after transfection, confluent cells were scratched with a P1000 sterile tip, held perpendicular to the plate. Media was removed and cells washed twice with pre-warmed PBS and once with pre-warmed selleck chem Dorsomorphin media. Fresh media was then added to the cells and they were photographed (0 hours) using the Olympus UC30 camera attached to an Olympus CKX41SF inverted microscope (Olympus, Tokyo, Japan). Cells were again washed at 24 and 48 hours post-scratch and photographed at these time-points also. All images were compared to the SCR control.

Quantitative Real-Time PCR miRNAs were extracted from the cells using the miRNeasy miRNA mini Kit (Qiagen GmbH, Crawley, West Sussex, UK) and quantitative Real-Time PCR was carried out as described above. Results Mutational Analysis Mutational analysis showed mutually exclusive KIT mutations in 18/73, PDGFRA in 11/73 cases and a BRAF mutation in a single case. The remaining 43 cases were WT for the exons tested in these genes. All clinical and genomic data are provided in Tables 1, ,2,2, ,33. Table 1 Adult Mutant GIST case demographics. Table 2 Adult WT case demographics. Table 3 Pediatric GIST case demographics. MicroRNA Profiling Unsupervised hierarchical clustering based on all miRNAs showed a separation into clusters A and B (Figure 1), with cluster B subdivided into B1 and B2.

Adult mutant cases are located in Clusters A and B1 and pediatric GISTs in Cluster B2, with adult WT GISTs dispersed amongst both adult mutant and pediatric WT cases (Figure 1). The clear split within the adult mutant cohort into clusters A and B1 is due to differential expression of forty-seven miRNAs located on chromosome 14q32.2 and 14q32.31. Following removal of the dominant 14q miRNA cluster from the heatmap, the split between adult mutant and pediatric WT GISTs is accentuated with the samples split into clusters C and D, such that adult mutant cases are in cluster C and pediatric WT cases in cluster D (Figure 2). Both these clusters can be further subdivided into C1, C2, D1 and D2 (Figure 2). The adult WT cases remain dispersed amongst both adult mutant and pediatric WT cases on this modified heatmap and the WT small bowel and retroperitoneal GISTs cluster tightly together in Cluster C2 with adult mutant cases (Figure 2).

In Cilengitide both these situations, the SDHB status underpins the clustering, such that the SDHB-immunopositive WT gastric, small bowel and retroperitoneal cases cluster with the adult mutant cases, while SDHB-immunonegative adult WT cases cluster with the pediatric WT cases. Sixteen miRNAs were found to be significantly differentially expressed between SDHB positive and SDHB negative cases.