Folate levels in mucosal tissue but not methylenetetrahydrofolate reductase polymorphisms are associated with gastric carcinogenesis

Table 1 Sequences of primers and probes for real-time PCR, and primers for MSP

Gene	Primer (forward) (5'-3')	Primer (reverse) (5'-3')	Probe	Genbank number
β-actin	CTG GCA CCC AGC ACA ATG	GGA CAG CGA GGC CAG GAT	ATC ATT GCT CCT CCT GAG	BC016045
p16INK4A	CAT AGA TGC CGC GGA AGG T	CAG AGC CTC TCT GGT TCT TTC AA	CCT CAG ACA TCC CCG	NM_058197
p21WAF1	CTG GAG ACT CTC AGG GTC GAA	GGA TTA GGG CTT CCT CTT GGA	ACG GCG GCA GAC CAG CAT GA	NM_078467
c-myc	ACA CCG CCC ACC ACC AG	CCA CAG AAA CAA CAT CGA TTT CTT	AGC GAC TCT GAG GAG G	V00568
hMLH1	GGC CAG CTA ATG CTA TCA AAG AG	CTT TAA CAA TCA CTT GAA TAC TTG TGG A	ATT GAG AAC TGT TTA GAT GCA	U07418
hMSH2	ATC CAA GGA GAA TGA TTG GTA TTT G	CAA AGA GAA TGT CTT CAA ACT GAG AGA	CAT ATA AGG CTT CTC CTG GC	U04045
p16INK4A(M)	TTA TTA GAG GGT GGG GCG GAT CGC	GAC CCC GAA CCG CGA CCG TAA		X94154
p16INK4A(U)	GGG GGA GAT TTA ATT TGG	CCC TCC TCT TTC TTC CTC		X94154
p21WAF1(M)	TGT AGT ACG CGA GGT TTC G	TCA ACT AAC GCA ACT CAA CG		NM_007592
p21WAF1(U)	TTT TTG TAG TAT GTG AGG TTT TGG	AAC ACA ACT CAA CAC AAC CCT A		NM_007592
c-myc (M)	TAG AAT TGG ATC GGG GTA AA	CGA CCG AAA ATC AAC GCG AAT		AF002859
c-myc (U)	TAG AAT TGG ATT GGG GTA AA	CCA ACC AAA AAT CAA CAT GAA T		AF002859
hMLH1 (M)	ACG TAG ACG TTT TAT TAG GGT CGC	CCT CAT CGT AAC TAC CCG CG		AB017806
hMLH1 (U)	TTT TGA TGT AGA TGT TTT ATT AGG GTT GT	ACC ACC TCA TCA TAA CTA CCC ACA		AB017806
hMSH2 (M)	TCG TGG TCG GAC GTC GTT C	CAA CGT CTC CTT CGA CTA CAC CG		AB006445
hMSH2 (U)	GGT TGT TGT GGT TGG ATG TTG TTT	CAA CTA CAA CAT CTC CTT CAA CTA CAC CA		AB006445

M: Methylation PCR primers; U: Unmethylation PCR primers.

Table 2 Folate level and transcription and methylation of c-myc

Methylationstatus	Transcription	Casenumber(n)	P1	Average folateconcentration(μg/L)	P2
Hypomethylated	Up-regulated	6		2.25 ± 1.23
	Unchanged	0	0.02		0.06
Methylated	Up-regulated	13		3.91 ± 2.01
	Unchanged	19

¹Transcription and DNA methylation of c-myc; ²Folate concentration and DNA methylation of c-myc.

Table 3 MTHFR gene polymorphisms and the transcription of tumor-related genes

Genes	MTHFR	Upregulation n (%)	Normal (n)	Downregulation n (%)	mRNA ratio of cancerous to noncancerous tissue (mean ± SD)	P
p16INK4A	CC	1	3	10 (71.43)	0.63 ± 1.05	0.84
	CT	1	8	10 (52.63)	0.7 ± 0.88	0.69
	TT	0	2	3 (60)	0.85 ± 0.96	-
	AA	1	9	16 (61.54)	0.57 ± 0.96	0.51
	AC	1	4	7 (58.33)	0.81 ± 1.15	-
p21WAF1	CC	1	7	6 (42.86)	1.06 ± 1.06	-
	CT	1	5	13 (68.42)	0.47 ± 0.69	0.062
	TT	0	2	3 (60)	0.55 ± 0.55	0.32
	AA	2	10	14 (53.85)	0.75 ± 0.85	0.57
	AC	0	4	8 (66.67)	0.58 ± 0.85	-
c-myc	CC	6 (42.86)	7	1	2.26 ± 2.17	0.048
	CT	11 (57.89)	6	2	4.32 ± 3.25	-
	TT	2 (40)	2	1	2.15 ± 1.8	0.17
	AA	15 (57.69)	8	3	3.84 ± 3.17	0.076
	AC	4 (33.33)	7	1	2.06 ± 1.64	-
hMLH1	CC	0	3	11 (78.57)	0.46 ± 0.67	0.053
	CT	1	8	10 (52.63)	0.57 ± 0.82	0.13
	TT	0	3	1 (25)	1.3 ± 1.28	-
	AA	2	10	14 (53.85)	0.72 ± 0.96	0.28
	AC	0	4	8 (66.67)	0.39 ± 0.55	-
hMSH2	CC	0	5	9 (64.29)	0.31 ± 0.32	-
	CT	1	10	8 (42.11)	0.88 ± 0.97	0.04
	TT	0	3	2 (40)	0.94 ± 1.12	0.064
	AA	1	13	12 (46.15)	0.8 ± 0.98	0.19
	AC	0	5	7 (58.33)	0.4 ± 0.42	-

Table 4 MTHFR polymorphisms of gastric cancerous tissue and CSG

Polymorphicsite	MTHFRgenotype	Canceroustissue n (%)	CSGn (%)	OR (95% CI)	P
C677T	CC	14 (36.84)	15 (44.12)	1 (reference)	-
	CT	19 (50.00)	11 (32.35)	1.85 (0.65-5.24)	0.24
	TT	5 (13.16)	8 (23.53)	0.67 (0.17-2.57)	0.55
A1298C
	AA	26 (68.42)	22 (64.71)	1 (reference)	-
	AC	12 (31.58)	11 (32.35)	0.92 (0.34-2.52)	0.87
	CC	0	1 (2.94)	-	-

Table 5 MTHFR polymorphism and biological characters of patients with gastric cancer (n)

Biological character		677CC	677CT	677TT	1298AA	1298AC
Degree of infiltration	Muscularis mucosae	2	2	1	3	2
	Muscular layer	1	3	2	5	1
	Serosa	7	9	1	12	5
	Out-serosa	4	5	1	6	4
Lymph node metastasis	Yes	7	11	2	14	6
	No	7	8	3	12	6
Tumor size	< 5 cm	9	7	2	11	7
	≥ 5 cm	5	12	3	15	5
TNM classification	I	2	2	1	3	2
	II	7	12	2	16	5
	III	5	5	2	7	5