Expression of Dnmt1, demethylase, MeCP2 and methylation of tumor-related genes in human gastric cancer

Table 1 Relationship between Dnmt1 and pathological change in patients with gastric cancer (case number, %)

	Subtotal	Dnmt1 transcription			P
		Up-regulated	Normal	Down-regulated
Total or subtotal	28 (100.0)	9 (32.14)	9 (32.14)	10 (35.72)
Age (yr) > 50	18 (64.30)	7 (38.89)	4 (22.22)	7 (38.89)	0.30
< 50	10 (35.70)	2 (20.00)	5 (50.00)	3 (30.00)
Size of tumor >5 cm	14 (50.00)	4 (28.57)	5 (35.71)	5 (35.71)	0.88
<5 cm	14 (50.00)	4 (28.57)	5 (35.71)	5 (35.71)
Borrmann II	6 (21.43)	2 (33.33)	0 (0.00)	4 (66.67)	0.34
III	15 (53.57)	5 (33.33)	6 (40.00)	4 (26.67)
IV	7 (25.00)	2 (28.57)	3 (42.86)	2 (28.57)
Lymph node Positive	21 (75.00)	6 (28.57)	8 (38.10)	7 (33.33)	0.49
invasion Negative	7 (25.00)	3 (42.86)	1 (14.29)	3 (42.86)

Table 2 Primer sequences and PCR conditions for RT-PCR, MSP and sequencing

Primers	Sense (5’→3’)	Antisense (5’→3’)	Product size (bp) PCR program	GenBank accession number
β -Actin	GGA GTC CTG TGG CAT CCA CG	CTA GAA GCA TTT GCG GTG GA	322 bp 94 °C 30 s, 60 °C 1 min, 72 °C 1 min, 27 cycles	BC023204
p16INK4A RT-PCR	CCC GCT TTC GTA GTT TTC AT	TTA TTT GAG CTT TGG TTC TG	355 bp 94 °C 1 min, 58 °C 1 min, 72 °C, 1 min, 35 cycles	L27211
c-myc RT-PCR	CCA ACA GGA GCT ATG ACC TC	CTC GGT CAC CAT CTC CAG CT	290 bp 94 °C 1 min, 52 °C 1 min, 72 °C, 1 min, 35 cycles	V00568
MeCP2 RT-PCR	ACT CCT CAG AAT ACA CCT TGC TT	TGA GGC CCT GGA GGT CCT	112 bp 95 °C 1 min, 50 °C 1 min, 72 °C, 1 min, 35 cycles	MCB, 2000; 20: 3316
mbd2 RT-PCR	AAC CCT GCT GTT TGC TTA AC	CGT ACT TGC TGT ACT CGC TCT TC	101 bp 94 °C 1 min, 60 °C 1 min, 72 °C, 1 min, 40 cycles	AF072242
p16INK4A M-MSP	TTA TTA GAG GGT GGG GCG GAT CGC	GAC CCC GAA CCG CGA CCG TAA	150 bp 95 °C 30 s, 65 °C 30 s, 72 °C, 1 min, 40 cycles	X94154
p16INK4A UM-MSP	TTA TTA GAG GGT GGG GTG GAT TGT	CAA CCC CAA ACC ACA ACC ATA A	151 bp 95 °C 30 s, 60 °C 30 s, 72 °C, 1 min, 40 cycles	X94154
c-myc M-MSP	TAG AAT TGG ATC GGG GTA AA	CGA CCG AAA ATC AAC GCG AAT	131 bp 95 °C 30 s, 56 °C 30 s, 72 °C, 1 min, 40 cycles	AF002859
c-myc UM-MSP	TAG AAT TGG ATT GGG GTA AA	CCA ACC AAA AAT CAA CAT GAA T	132 bp 95 °C 30 s, 56 °C 30 s, 72 °C, 1 min, 40 cycles	AF002859
hMSH2 sequencing 1st round	TGT TTA GAA AGA AAA AGG GA	AAA CCT CCT CAC CTC CT	94 °C 1 min, 55 °C 1 min, 72 °C, 1 min, 35 cycles	AB006445
hMSH2 sequencing 2nd round	AAA TAT TGG GAG GAG GAG GA	ACC CAC TAA ACT ATT TCC CA	327 bp 94 °C1 min,, 55 °C 1 min, 72 °C, 1 min, 35 cycles	AB006445

Table 3 Sequence of primers and probes for real-time PCR

Gene	Sense (5’→3’)	Antisense (5’→3’)	Probe	GenBank No
Dnmt1	GCA CCT CAT TTG CCG AAT ACA	TCT CCT GCA TCA GCC CAA ATA	AGT CCC GAG TAT GCG C	XM_017218
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	HSU04045
β -actin	CTG GCA CCC AGC ACA ATG	GGA CAG CGA GGC CAG GAT	ATC ATT GCT CCT CCT GAG	BC016045