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

Table 1 Sex, age and disease duration of cases of each stage (mean ± SD)

Stage	n	M	F	Age, yr	Disease duration (yr)
NGM without H. pylori infection	25	14	11	54.12 ± 8.21 (45-60)	0.85 ± 0.64 (0.12-1.23)
NAG with H. pylori infection	24	13	11	56.44 ± 11.29 (47-65)	0.85 ± 0.35 (0.37-1.03)
CAG with H. pylori infection	25	11	14	55.90 ± 7.80 (45-66)	0.73 ± 0.24 (0.13-0.90)
IM with H. pylori infection	28	12	16	52.16 ± 8.59 (48-68)	0.90 ± 0.65 (0.47-1.04)
DYS with H. pylori infection	24	12	12	54.05 ± 7.36 (48-69)	0.73 ± 0.57 (0.56-1.15)
GC with H. pylori infection	30	17	13	55.43 ± 10.33 (46-73)	0.60 ± 0.46 (0.10-1.04)

NGM: Normal gastric mucosa; NAG: Non-atrophic gastritis; CAG: Chronic atrophic gastritis; IM: Intestinal metaplasia; DYS: Dysplasia; GC: Gastric cancer.

Table 2 Cx32 and Cx43 primer sequences, amplified fragment size and annealing temperature

Method	Gene			Primer sequence (5’→3’)	Amplified fragment size (bp)	Annealing temperature (°C)
Real-time RT-PCR	Cx32		F:	ATGAACTGGACAGGTTTGTAC	302	56
			R:	ATGTGTTGCTGGTGCAGCCA
	Cx43		F:	TGCAGCAGTCTGCCTTTCGTTG	219	56
			R:	CCATCAGTTTGGGCAACCTTG
	β-actin		F:	TGGACTTCGCAGCACAGCAGATGG	289	56
			R:	ATCTCCTTCTGCATCCTGTCG
MSP	Cx32	M	F:	GGGGCGGGTGCGGCGAT	245	64
			R:	CTCCGCGCCTACGTCCC
		U	F:	GGGGTGGGTGTGGTGAT	245	64
			R:	CTCCACACCTACATCCCAA
	Cx43	M	F:	AAATTGTAATATTTGGGTTTCAGCGC	156	58
			R:	AATAACGCCATCTCTACTCACCG
		U	F:	TTTTAAAATTGTAATATTTGGGTTTCAGTGT	161	56
			R:	AATAACACCATCTCTACTCACCACA
BSP	Cx32		F:	GGTTATTTTTTTGGTGGGGTTATG	313	58
			R:	ACCCAAACAAATCCCCTATAATCTC
	Cx43		F:	TGTTTTTTAAAATTGTAATATTTGGGTTTA	377	56
			R:	AAAAACAAACTCATCTAACCTTCCTATTC
MassArray	Cx32		F:	CAGTTT CAGCAGTTTTTGGGTTTTTTGG	484	60
			R:	TAACTCCCTATCCCCTAACTCCTTA
	Cx43		F:	ATGTTTTTGCAGGTTGGATCAGGAAAT	447	60
			R:	ACCAACAAATAAAAACAAAATTATTCC

Table 3 Cx32 and Cx43 CpG loci detected by bisulfite polymerase chain reaction sequencing

Cx32	CpG	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
	bp	28	34	37	45	49	83	86	120	132	155	227	231	247	253	255
		○	○	○	○	●	●	●	●	●	●	●	●	●	●	●
Cx43	CpG	1	2	3	4	5	6	7	8	9	10	11	12
	bp	32	34	37	44	71	127	134	143	159	190	198	280
		○	○	○	○	●	●	●	●	●	●	●	●

“●” denotes the detected CpG site; “○”denotes the undetected CpG site.

Table 4 Cx32 and Cx43 promoter methylation status at different gastric carcinogenesis stages with Helicobacter pylori infection by bisulfite polymerase chain reaction sequencing method

	n	Cx32 (%)	Cx43 (%)
NGM without H. pylori infection	7	60.1 ± 5.9	75.5 ± 4.3
NAG with H. pylori infection	6	67.3 ± 4.9	82.9 ± 6.3
CAG with H. pylori infection	7	74.5 ± 7.5a	87.1 ± 5.4a
IM with H. pylori infection	7	84.2 ± 6.8ace	90.5 ± 9.3ac
DYS with H. pylori infection	6	82.3 ± 6.0ac	91.6 ± 8.3ac
GC with H. pylori infection	9	85.3 ± 9.7ace	92.0 ± 7.1ac

^aP < 0.05 vs NM;

^cP < 0.05 vs NAG;

^eP < 0.05 vs CAG. NGM: Normal gastric mucosa; NAG: Non-atrophic gastritis; CAG: Chronic atrophic gastritis; IM: Intestinal metaplasia; DYS: Dysplasia; GC: Gastric cancer.

Table 5 Methylation levels (%) of Cx32 and Cx43 CpG islands at gastric different stages with Helicobacter pylori infection detected by MassArray method

	Cx32 (9 loci)	Cx43 (8 loci)
NGM without H. pylori infection	55.0 ± 14.9	77.9 ± 16.8
NAG with H. pylori infection	61.2 ± 17.9	86.0 ± 12.5
CAG with H. pylori infection	69.9 ± 16.2	88.6 ± 9.9
IM with H. pylori infection	72.4 ± 19.2a	89.1 ± 9.6
DYS with H. pylori infection	72.6 ± 19.2a	90.0 ± 9.3a
GC with H. pylori infection	76.0 ± 20.1a	91.6 ± 8.2a

^aP < 0.05 vs NGM. NGM: Normal gastric mucosa; NAG: Non-atrophic gastritis; CAG: Chronic atrophic gastritis; IM: Intestinal metaplasia; DYS: Dysplasia; GC: Gastric cancer.