We explain the lack of a perfect correlation between the methylation status of the 14q region and miRNA expression by likely admixture of DNA from non-tumor tissue elements retaining the normally imprinted arrangement. Our hypothesis does not hold true for the pediatric WT cases, with the majority of cases tested Ruxolitinib buy showing normal methylation patterns, suggesting that some other mechanism must underlie the down-regulation of the miRNAs in this region in this setting. Further investigation is required to elucidate this. Many miRNAs are categorised into clusters, often located at fragile sites [44]. The forty-seven miRNAs located on 14q32 represent one of the largest clusters identified to-date [45] and have been found down-regulated or silenced in many cancers including gliomas [45] and epithelial ovarian cancers [46], suggesting a possible tumor suppressor role for the miRNAs located in this cluster.

Few of the 14q32 miRNAs have been studied in vitro with target validation. Of those differentially expressed in our study, 14q32 miRNAs having confirmed targets include miR-127 (located within a CpG island and silenced in many cancers) targeting BCL-6 [47], miR-154 targeting CCND2 [48], miR-433 targeting HDAC6 [49], miR-485-3p targeting NF-YB [50] and miR-539 targeting HLCS [51]. Using target prediction algorithms, including TargetScan, [18]�C[20], it emerges that these miRNAs may target genes of known importance in GIST cell biology including KIT, PDGFRA IGF1R, NF1, MAPK1, SDH and KRAS [9], [14], [28], however further investigation is required to confirm this as well as other potentially important gene targets.

Recently miR-494 was shown to target KIT in the GIST882 cell line [52]. Up-regulation of miR-494 in GIST882 cells reduced the expression of KIT and its downstream signalling, increased apoptosis and inhibited cell growth, suggesting the possible importance of loss of miR-494 and perhaps indeed all 47 miRNAs on 14q32 in GIST progression [52]. On the heatmaps (Figures 1; 2), the pediatric cases consistently cluster separately from the adult mutant cases. As described, our functional assays suggest that the differentially expressed miRNAs producing this clustering indeed have biological effects on GIST cell lines. The miRNAs chosen here have proven targets in other neoplasms. miR-34c-5p targets E2F3, MYCN, Bcl-2 and c-Met [53], Carfilzomib [54], while miR-185 targets RhoA and Cdc42 [55]. Ectopic expression of miR-34c-5p in cervical cancer cells inhibited proliferation and anchorage independent growth, while ectopic expression of miR-185 in colorectal cancer cells also inhibited proliferation, induced G1 cell cycle arrest and apoptosis, and blocked invasion, suggesting tumor suppressive roles for both these miRNAs [53]�C[55].