icated in the pathogenesis of GIST. PDGFRA mRNA contains putative miR-34a binding sites in its 3′ UTR . Reporter assays using a luciferase vector containing wild-type miR-34a binding sites revealed that cotransfection of a miR-34a mimic markedly downregulated luciferase activity in GIST-T1 cells. Such downregulation was not observed when cells were PD-1/PD-L1 inhibitor 2 transfected with a luciferase vector containing mutant miR-34a binding sites. Suppression of PDGFRA expression by miR-34a was further confirmed by quantitative RT-PCR using GIST-T1 cells. In addition, PDGFRA knockdown using a specific siRNA suppressed the viability of GIST-T1 cells, suggesting the tumor suppressive effect of miR-34a may be mediated at least in part through targeting PDGFRA. We also performed a gene expression microarray analysis using GIST-T1 cells transfected with a miR-335 mimic, and found that 1,095 probes representing 853 unique genes were downregulated. Gene ontology analysis showed that “D-aspartate transport,” “neuronal action potential” and “epidermis development” genes were enriched among the downregulated 9 / 17 Screen for miRNA Gene Methylation in GISTs Fig 4. Functional analysis of miR-34a and miR-335. Cell viability assays using GIST-T1 cells transfected with miR-34a or miR-335 mimics or a negative control. Cell viabilities were determined 72 h after transfection. Shown are the means of 8 replications; error bars represent standard deviations; P values were determined using Student’s t test. Wound healing assay using GIST-T1 cells transfected with a miRNA mimic or a negative control. Shown on the right are the means of 4 replications; error bars represent standard deviations; P values were determined using Student’s t test. Cell migration and Matrigel invasion assays using GIST-T1 cells transfected with a miRNA mimic or a negative control. Arrowheads indicate migrating or invading cells. Shown on the right are the means of 5 random microscopic fields per membrane; error bars represent standard deviations; P values were determined using Student’s t test. doi:10.1371/journal.pone.0133754.g004 genes. Microarray analysis also revealed that 16 predicted target genes, including EIF5A2, ZMPSTE24 and MAT2B, were downregulated by miR-335 in GIST-T1 cells. Discussion In this study, we tried to identify miRNA genes that are potentially inactivated through an epigenetic mechanism in GIST cells. Despite applying a stringent criterion in the first step of our screening, we found 61 miRNAs to be 10 / 17 Screen for miRNA PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19747578 Gene Methylation in GISTs Fig 5. Downregulation of predicted miR-34a target genes in GIST-T1 cells. Venn PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19748727 diagram for genes downregulated by ectopic miR-34a expression in GIST-T1 cells and predicted miR-34a target genes. Of the 49 downregulated target genes, the top 10 genes are listed on the right. Expression levels and fold-changes are also indicated. Putative miR-34a binding sites in the 3′ untranslated region of PDGFRA. Mutant binding sites used for the luciferase assay are shown in red. Reporter assay results using a luciferase vector containing the wild-type PDGFRA 3′ UTR or the mutant 3′ UTR in GIST-T1 cells cotransfected a miR-34a mimic or a negative control. Shown are means of 4 replications; error bars represent 11 / 17 Screen for miRNA Gene Methylation in GISTs standard deviations; the P value was determined using Student’s t test. Quantitative RT-PCR of PDGFRA in GIST-T1 cells transfected with a miR-34a mimic or a negative control. 
Quantitative RT-PCR
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