Small RNA sequencing-based miRNA profiling and functional analysis in gastrointestinal stromal tumors (GISTs)

Abstract

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional regulation of gene expression. Deregulated miRNA profiles and their contribution to carcinogenesis has been observed in different types of cancer. However, their involvement in pathogenesis of rare gastrointestinal stromal tumors (GISTs) is not yet fully understood. Therefore, the aim of this study was to determine highly deregulated miRNAs in GIST and to investigate their possible involvement in GIST pathogenesis. MiRNA expression profile was determined using small RNA-seq approach in paired tumor and adjacent tissue samples of 15 GIST patients and validated by Taq-Man low-density array in a larger sample group of 40 patients. Potential targets of deregulated miRNAs were predicted using TargetScan Release 7.1. Further transfections with miRNA mimics and functional analysis was performed in GIST-T1 cell line. Changes in target gene expression were detected using TaqMan primers and probes, protein expression analysis was performed using Western Blot technique. Alterations in cell viability and migration rates were evaluated by MTT and Wound Healing Assays. Statistical analysis was performed using the computing environment R. Sequencing data showed distinct miRNA expression profiles between tumor and adjacent tissue samples. In a further validation step, expression levels of 19 miRNAs showed significant differential expression in the same direction as in the sequencing data (Bonf. adj. p < 0.01; FC > 2). MiRNAs, potentially targeting genes involved in cancer associated signaling pathways, were selected (hsa-miR-375, hsa-miR-200b-3p, hsa-miR-490-3p) for target gene expression and functional analysis in GIST-T1 cell line. Increased amounts of hsa-miR-375 significantly reduced expression of its predicted target gene KIT (FC = 1.8, p < 0.05), however KIT protein expression remained unchanged. Overexpression of hsa-miR-200b-3p significantly reduced EGFR and ETV1 gene expression