Fig 1.
Identification of epigenetically silenced miRNAs in GIST-T1 cells.
(A) Workflow of the screen to identify epigenetically silenced miRNAs. (B) Summarized TaqMan array results for 22 candidate miRNAs in GIST-T1 cells treated with or without 5-aza-dC plus PBA. Expression levels were normalized to that of U6 snRNA expression.
Fig 2.
Analysis of CGI methylation at predicted transcription start sites of miRNA genes.
(A) Representative examples of miRNA gene structures. Host genes encoding miR-34a and miR-335 are shown. CGIs and pre-miRNA regions are shown below. (B) Levels of CGI methylation in the selected miRNA genes were analyzed using bisulfite pyrosequencing in GIST-T1 cells. (C) Results of bisulfite pyrosequencing of miR-335 and miR-34a in GIST-T1 cells.
Fig 3.
Analysis of miRNA gene methylation in primary GIST specimens.
(A) Summarized results showing the methylation levels of selected miRNAs in primary tumors analyzed using bisulfite pyrosequencing. (B) Representative results of bisulfite pyrosequencing of miR-335 and miR-34a in primary tumors. (C) Summarized results showing bisulfite pyrosequencing of miR-335 and miR-34a in primary GIST specimens.
Table 1.
Correlation between methylation of miRNA genes and the clinicopathological features of GIST patients.
Fig 4.
Functional analysis of miR-34a and miR-335.
(A) 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. (B) 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. (C,D) Cell migration (C) and Matrigel invasion (D) 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.
Fig 5.
Downregulation of predicted miR-34a target genes in GIST-T1 cells.
(A) Venn diagram for genes downregulated by ectopic miR-34a expression in GIST-T1 cells (>1.5-fold) 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. (B) Putative miR-34a binding sites in the 3’ untranslated region (UTR) of PDGFRA. Mutant binding sites used for the luciferase assay are shown in red. (C) Reporter assay results using a luciferase vector containing the wild-type PDGFRA 3’ UTR (PDGFRA) or the mutant 3’ UTR (PDGFRA-mut) in GIST-T1 cells cotransfected a miR-34a mimic or a negative control. Shown are means of 4 replications; error bars represent standard deviations; the P value was determined using Student’s t test. (D) Quantitative RT-PCR of PDGFRA in GIST-T1 cells transfected with a miR-34a mimic or a negative control. (E) Quantitative RT-PCR of PDGFRA in GIST-T1 cells transfected with a siRNA targeting PDGFRA (siPDGFRA) or a control siRNA (siCONT). (F) Cell viability assays using GIST-T1 cells transfected with siCONT or siPDGFRA. Cell viabilities were determined 72 h after transfection. Shown are means of 8 replications; error bars represent standard deviations; the P value was determined using Student’s t test.