Connexin 32 and 43 promoter methylation in Helicobacter pylori-associated gastric tumorigenesis

Figure 1 Location of primers for polymerase chain reaction for methylation detection.

Figure 2 Real-time polymerase chain reaction results for Cx32 and Cx43 mRNAs. A: The amplification curve and melting curve of the real-time polymerase chain reaction; B: The relative expression of Cx32 and Cx43 mRNAs at different stages. ^aP < 0.05 vs NGM; ^cP < 0.05 vs NAG; ^eP < 0.05 vs CAG; ^gP < 0.05 vs IM; ⁱP < 0.05 vs DYS. NGM: Normal gastric mucosa; NAG: Non-atrophic gastritis; CAG: Chronic atrophic gastritis; IM: Intestinal metaplasia; DYS: Dysplasia; GC: Gastric cancer.

Figure 3 Methylation-specific polymerase chain reaction results for Cx32 and Cx43 promoters at different gastric carcinogenesis stages with Helicobacter pylori infection. A: The agarose gel electrophoresis of the methylation-specific polymerase chain reaction (MSP) bands. Marker: 50bp ladder; M: Methylated; U: Unmethylated; MP: Methylation positive control; UP: Unmethylated positive control; DW: Negative control. B: The methylation levels of Cx32 and Cx43 promoters at different stages by MSP method. ^aP < 0.05 vs NGM; ^cP < 0.05 vs NAG; ^eP < 0.05 vs CAG; ^gP < 0.05 vs IM; ⁱP < 0.05 vs DYS. NGM: Normal gastric mucosa; NAG: Non-atrophic gastritis; CAG: Chronic atrophic gastritis; IM: Intestinal metaplasia; DYS: Dysplasia; GC: Gastric cancer.

Figure 4 Screenshots of bisulfite polymerase chain reaction sequencing of the Cx32 and Cx43 promoter CpG islands. The upper two show the 10^thCx32 CpG site (155bp), with the C/(C+T) ratio of 60% in NM and 80% at GC stage. The lower two shows the 10^th and 11^thCx43 CpG sites (190bp, 198bp). Dark arrow indicates the peak of the sulfonated methyl-CpG site still as C; red arrow indicates the sulfonated non-methylated CpG site changed as T.

Figure 5 Methylation levels of Cx32 and Cx43 promoters at different gastric carcinogenesis stages with Helicobacter pylori infection by MassArray method. The validated length for Cx32 gene was 484 bp, containing a total of 18 CpG sites (15 detected); the validated length for Cx43 gene was 447 bp, containing a total of 12 CpG sites (11 detected).