Hypermethylation of tumor suppressor and tumor-related genes in neoplastic and non-neoplastic gastric epithelia

Figure 1 Methylation status of the RUNX3 CpG island in primary gastric cancer and non-neoplastic gastric mucosa. A: RUNX3 CpG island and analyzed regions (No. 1-10). CpG sites are shown as vertical bars. The transcription start site (TS) is located within region No. 7. B: Summary of methylation and expression status of RUNX3 in gastric cancer cell lines. C: Percentage of methylation at multiple regions (No. 1-10) of the RUNX3 CpG island in primary gastric cancer and non-neoplastic gastric mucosa.

Figure 2 A schematic model of methylation spreading and gene silencing. A: Status of completely unmethylated CpGs with gene transcription (arrow); B: Appearance of methylated CpGs at the edge of CpG islands with gene transcription (arrow); C: Gene silencing by CpG methylation at the transcription start site.

Figure 3 Locations of primers (arrows) and probes (boxes) used in microarray analysis, transcription start sites (closed triangles), and start codons (open triangles). Vertical bars indicate CpGs.

Figure 4 Standardization for DNA microarray analysis. A: Microarray images of control DNAs with methylation rates of 0%, 25%, 50%, 75%, and 100%. M: Methylated sequence probe; U: Unmethylated sequence probe. Three replicates of the experiment were used to create standard curves. B: Standard curves were obtained for LOX: y = -49.256x² + 150.4x - 1.0847, R² = 0.9985; p16: y = 24.965x² + 81.528x - 2.9098, R² = 0.9617; RUNX3: y = 44.245x² + 61.71x - 2.8558, R² = 0.984; and TIG1: y = -20.932x² + 133.9x - 11.113, R² = 0.9852.

Figure 5 Methylation rates of tumor suppressor genes in primary gastric cancers (gray bar) and non-neoplastic gastric epithelia (white bar).

Figure 6 Regression curves. LOX: y = 0.013x² - 0.6184x + 4.0512, R² = 0.5728, P < 0.001; p16: y = 0.0107x² - 0.6055x + 5.2943, R² = 0.7891, P < 0.00001; RUNX3: y = 0.0182x² - 1.2234x + 11.566, R² = 0.5595, P < 0.001; and TIG1: y = 0.0068x² - 0.3586x + 2.4306, R² = 0.4670, P < 0.01.