Abstract
BackgroundDysregulation of microRNA (miRNA) has been implicated in gastrointestinal stromal tumors (GISTs) but the mechanism is not fully understood. In this study, we aimed to explore the involvement of epigenetic alteration of miRNA genes in GISTs.MethodsGIST-T1 cells were treated with 5-aza-2’-deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which miRNA expression profiles were analyzed using TaqMan miRNA arrays. DNA methylation was then analyzed using bisulfite pyrosequencing. The functions of miRNAs were examined using MTT assays, wound-healing assays, Boyden chamber assays and Matrigel invasion assays. Gene expression microarrays were analyzed to assess effect of ectopic miRNA expression in GIST-T1 cells.ResultsOf the 754 miRNAs analyzed, 61 were significantly upregulated in GIST-T1 cells treated with 5-aza-dC plus PBA. Among those, 21 miRNA genes were associated with an upstream CpG island (CGI), and the CGIs of miR-34a and miR-335 were frequently methylated in GIST-T1 cells and primary GIST specimens. Transfection of miR-34a or miR-335 mimic molecules into GIST-T1 cells suppressed cell proliferation, and miR-34a also inhibited migration and invasion by GIST-T1 cells. Moreover, miR-34a downregulated a number of predicted target genes, including PDGFRA. RNA interference-mediated knockdown of PDGFRA in GIST-T1 cells suppressed cell proliferation, suggesting the tumor suppressive effect of miR-34a is mediated, at least in part, through targeting PDGFRA.ConclusionsOur results suggest that miR-34a and miR-335 are candidate tumor suppressive miRNAs in GISTs, and that they are frequent targets of epigenetic silencing in GISTs.
Highlights
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract [1]
21 miRNA genes were associated with an upstream CpG island (CGI), and the CGIs of miR-34a and miR-335 were frequently methylated in GIST-T1 cells and primary GIST specimens
Our results suggest that miR-34a and miR-335 are candidate tumor suppressive miRNAs in GISTs, and that they are frequent targets of epigenetic silencing in GISTs
Summary
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract [1]. Approximately onethird of GISTs with no KIT mutations carry mutations in PDGFRA, which encodes plateletderived growth factor receptor α. In addition to KIT and PDGFRA mutations, a majority of GISTs acquire other genetic and epigenetic abnormalities during their malignant progression. Recent array CGH analyses identified a number of chromosomal imbalances that could be relevant to the pathogenesis of GISTs [2, 3]. In addition to genetic alterations, aberrant DNA methylation has been implicated in the development of GISTs. We previously showed that hypomethylation of repetitive sequences, including LINE-1, correlates with increased chromosomal aberration and GIST malignancy [4], and a recent genome-wide DNA methylation analysis revealed that hypermethylation of three genes (REC8, PAX3 and p16) is strongly associated with aggressive clinical behavior and an unfavorable prognosis [5]
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