Promoter methylation and mRNA expression of DKK-3 and WIF-1 in hepatocellular carcinoma

Table 1 Methylation-specific PCR, real-time PCR primer, probe sequence and annealing temperature

Gene	Primer purpose	Primer sequence (5'--3')	Annealing temperature (°C)	Product size (bp)
DKK-3	MSP-M	F: GGGGCGGGCGGCGGGGC	58	120
		R: ACATCTCCGCTCTACGCCCG
	MSP-U	F: TTAGGGGTGGGTGGTGGGGT	56	126
		R: CTACATCTCCACTCTACACCCA
	Real time RT-PCR	F: GTAAGTTTCCCCTCTGGCTTG	60	90
		R: AAGCACCAGACTGTGAAGCCT
		Probe: FAM+AGGTGTTGTGCATTTGTTCAGCTCCC+TAMRA
WIF-1	MSP-M	F: CGTTTTATTGGGCGTATCGT	57	145
		R: ACTAACGCGAACGAAATACGA
	MSP-U	F: GGGTGTTTTATTGGGTGTATTGT	52	154
		R: AAAAAAACTAACACAAACAAAATACAAAC
	Real time RT-PCR	F: TCCAAACACCTCAAAATGCTATC	60	119
		R: GAACCCATCAGGACACTCGC
		Probe: FAM+ACAAGCTGAGTGCCCAGGCGG+TAMRA
GAPDH	Real time RT-PCR	F: CCACTCCTCCACCTTTGAC	60	102
		R: ACCCTGTTGCTGTAGCCA
		Probe: FAM+TTGCCCTCAACGACCACTTTGTC+TAMRA

M: Methylation; U: Unmethylation; F: Sense primer; R: Antisense primer.

Table 2 Methylation level of DKK-3 and WIF-1 in two tissues (mean ± SE, %)

Methylation level	DKK-3	WIF-1
HCC tissues	37.61 ± 4.26	29.70 ± 3.24
Adjacent non-cancerous tissues	15.96 ± 3.91	18.34 ± 4.02
	P < 0.05	P < 0.05

Table 3 Methylation and clinical data of DKK-3 genes in HCC and adjacent non-cancerous tissues

Clinical data	Methylation in HCC			Methylation in adjacent non-cancerous tissues
	Negative	Positive	P	Negative	Positive	P
Sex
Male	12	15	1.000	21	6	1.000
Female	3	3		5	1
Age (yr)
< 60	13	7	0.011	17	3	0.393
≥ 60	2	11		9	4
Child
A	12	17	0.308	23	6	1.000
B	3	1		3	1
AFP (ng/mL)
< 20	7	10	0.308	15	3	0.674
≥ 20	8	8		11	4
Number of tumor
Single	11	13	1.000	20	4	0.358
Multiple	4	5		6	3
Tumor diameter (cm)
< 3	2	3	1.000	3	2	1.000
≥ 3	13	15		23	5
HBsAg
(+)	15	14	0.108	23	6	1.000
(-)	0	4		3	1
Hepatocirrhosis
(+)	15	10	0.004	20	5	1.000
(-)	0	8		6	2
False capsule
(+)	14	14	0.346	23	5	0.282
(-)	1	4		3	2
Tumor thrombus
(+)	1	2	1.000	3	0	1.000
(-)	14	16		23	7
Edmonson stage
I/II	13	15	1.000	22	6	1.000
III	2	3		4	1

Table 4 Methylation and clinical data of WIF-1 genes in HCC and adjacent non-cancerous tissues

Clinical data	Methylation in HCC			Methylation in adjacent non-cancerous tissues
	Negative	Positive	P	Negative	Positive	P
Sex
Male	17	10	0.659	23	4	1.000
Female	3	3		5	1
Age (yr)
< 60	7	5	1.000	17	3	1.000
≥ 60	13	8		11	2
Child
A	17	12	1.000	24	5	1.000
B	3	1		4	0
AFP (ng/mL)
< 20	11	8	1.000	16	3	1.000
≥ 20	9	5		12	2
Number of tumor
Single	14	10	1.000	21	3	0.597
Multiple	6	3		7	2
Tumor diameter (cm)
< 3	2	3	1.000	5	0	0.569
≥ 3	18	10		23	5
HBsAg
(+)	20	9	0.017	24	5	1.000
(-)	0	4		4	0
Hepatocirrhosis
(+)	18	7	0.035	21	4	1.000
(-)	2	6		7	1
False capsule
(+)	18	10	0.360	24	4	1.000
(-)	2	3		4	1
Tumor thrombus
(+)	1	2	0.547	2	1	0.400
(-)	19	11		26	4
Edmonson stage
I/II	16	12	0.625	23	5	0.569
III	4	1		5	0

Table 5 mRNA expression and clinical data of DKK-3 in HCC and adjacent non-cancerous tissues (mean ± SE)

Clinical data	Case	The mRNA expression of DKK-3 in HCC	t	P	The mRNA expression of DKK-3 in adjacent non-cancerous tissues	t	P
Sex
Male	27	0.776 ± 0.346	0.153	0.880	0.779 ± 0.319	0.888	0.404
Female	6	0.761 ± 0.168			0.913 ± 0.338
Age (yr)
< 60	20	0.756 ± 0.356	0.334	0.741	0.819 ± 0.341	0.195	0.847
≥ 60	13	0.795 ± 0.285			0.840 ± 0.274
Child
A	29	0.761 ± 0.331	0.789	0.466	0.829 ± 0.329	0.171	0.869
B	4	0.863 ± 0.225			0.813 ± 0.155
AFP (ng/mL)
< 20	17	0.716 ± 0.287	1.069	0.294	0.840 ± 0.354	0.124	0.902
≥ 20	16	0.835 ± 0.384			0.826 ± 0.281
Number of tumor
Single	24	0.740 ± 0.315	0.971	0.348	0.823 ± 0.298	0.262	0.797
Multiple	9	0.860 ± 0.329			0.860 ± 0.377
Tumor diameter (cm)
< 3	4	0.908 ± 0.300	0.943	0.399	0.792 ± 0.352	0.290	0.780
≥ 3	29	0.755 ± 0.322			0.841 ± 0.315
HBsAg
(+)	29	0.660 ± 0.233	0.984	0.372	0.817 ± 0.304	0.639	0.563
(-)	4	0.789 ± 0.329			0.955 ± 0.418
Hepatocirrhosis
(+)	25	0.780 ± 0.319	0.046	0.964	0.761 ± 0.268	2.157	0.057
(-)	8	0.774 ± 0.365			1.060 ± 0.361
False capsule
(+)	28	0.757 ± 0.326	1.750	0.132	0.819 ± 0.307	0.790	0.466
(-)	5	1.014 ± 0.299			0.966 ± 0.395
Tumor thrombus
(+)	3	0.770 ± 0.330	0.738	0.519	0.811 ± 0.307	1.088	0.379
(-)	30	0.890 ± 0.260			1.057 ± 0.379
Edmonson stage
I/II	28	0.807 ± 0.335	3.380	0.020	0.820 ± 0.317	0.564	0.596
III	5	0.586 ± 0.350			0.910 ± 0.333

Table 6 mRNA expression and clinical data of WIF-1 in HCC and adjacent non-cancerous tissues (mean ± SE)

Clinical data	Case	The mRNA expression of WIF-1 in HCC	t	P	The mRNA expression of WIF-1 in adjacent non-cancerous tissues	t	P
Sex
Male	27	0.882 ± 0.542	0.942	0.365	0.828 ± 0.320	0.286	0.783
Female	6	0.720 ± 0.336			0.787 ± 0.318
Age (yr)
< 60	12	0.767 ± 0.397	1.092	0.289	0.793 ± 0.247	0.551	0.589
≥ 60	21	0.985 ± 0.643			0.862 ± 0.407
Child
A	29	0.854 ± 0.543	0.067	0.948	0.793 ± 0.309	1.243	0.286
B	4	0.845 ± 0.171			1.015 ± 0.337
AFP (ng/mL)
< 20	19	0.873 ± 0.516	0.231	0.819	0.828 ± 0.377	0.148	0.883
≥ 20	14	0.831 ± 0.519			0.812 ± 0.247
Number of tumor
Single	24	0.927 ± 0.532	1.292	0.212	0.801 ± 0.288	0.496	0.630
Multiple	9	0.704 ± 0.408			0.872 ± 0.393
Tumor diameter (cm)
< 3	5	1.002 ± 0.750	0.507	0.636	0.796 ± 0.415	0.147	0.889
≥ 3	28	0.826 ± 0.469			0.825 ± 0.304
HBsAg
(+)	29	0.875 ± 0.527	0.155	0.886	0.838 ± 0.312	0.754	0.497
(-)	4	0.820 ± 0.681			0.695 ± 0.360
Hepatocirrhosis
(+)	25	0.840 ± 0.523	0.260	0.797	0.810 ± 0.322	0.331	0.746
(-)	8	0.893 ± 0.486			0.853 ± 0.314
False capsule
(+)	28	0.860 ± 0.490	0.152	0.886	0.816 ± 0.296	0.144	0.892
(-)	5	0.812 ± 0.676			0.846 ± 0.454
Tumor thrombus
(+)	3	0.815 ± 0.505	1.392	0.276	0.806 ± 0.319	0.840	0.474
(-)	30	1.223 ± 0.481			0.960 ± 0.300
Edmonson stage
I/II	28	0.910 ± 0.517	1.005	0.083	0.821 ± 0.308	0.141	0.894
III	5	0.532 ± 0.360			0.848 ± 0.401