Aberrant methylation of SPARC in human hepatocellular carcinoma and its clinical implication

Table 1 Primer sequences for polymerase chain reaction

Gene	Primer sequences (forward/reverse 5’-3’)	Accession No.	Location to transcription start	Product size (bp)	Annealing temperature (°C)
SPARC methylation	GAGAGCGCGTTTTGTTTGTC	NM_003118.2	+52 to +71	112	54
	AACGACGTAAACGAAAATATCG		+142 to +163
SPARC unmethylation	TTTTTTAGATTGTTTGGAGAGTG	NM_003118.2	+36 to +58	132	59
	AACTAACAACATAAACAAAAATATC		+143 to +167
SPARC BS	GATAGAGATAGTTTTGGTTATGGGA	NM_003118.2	-119 to -95	401	55
	CCACCTTCTAAAAAACA ACAAAC		+260 to +282
SPARC mRNA	CGCATGCGGGACTGGCTCAA	NM_003118.2	+601 to +620	148	60
	GCTCCACGGGG TGGTC TCCT		+729 to +748
GAPDH mRNA	GGGCATCCTGGGCTACACTGA	NM_002046.3	+915 to +935	143	58
	CAAATTCGTTGTCATACCAGGAAATG		+1032 to +1057

SPARC: Secreted protein acidic and rich in cysteine; BS: Bisulfite sequencing; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Table 2 Methylation frequencies of secreted protein acidic and rich in cysteine in 60 cases

	SPARC methylation status
Tissue	Methylated (%)	Unmethylated (%)	P value
Tumorous	45 (75.00)	15 (25.00)	< 0.001
Nontumorous	7 (11.67)	53 (88.33)

SPARC: Secreted protein acidic and rich in cysteine.

Table 3 Protein expression frequencies in 23 pairs of samples

		Protein expression
Tissue	n	Positive (%)	Negative (%)	P value
Tumorous	23	12 (52.2)	11 (47.8)	0.552
Nontumorous	23	14 (60.9)	9 (39.1)

Table 4 Association of secreted protein acidic and rich in cysteine methylation with protein expression

		Protein expression
SPARC	n	Positive (%)	Negative (%)	P value
Methylated	14	6 (42.9)	8 (57.1)	0.216
Unmethylated	32	20 (62.5)	12 (37.5)

SPARC: Secreted protein acidic and rich in cysteine.

Table 5 Correlation between methylation status and clinicopathological data

Parameters	n	Methylated	Unmethylated	P value
Age (yr)				0.766
> 53	30	23	7
≤ 53	30	22	8
Gender				0.835
Male	51	38	13
Female	9	7	2
Tumor size (cm)				1.000
≤ 5	20	15	5
> 5	40	30	10
Virus infection				0.661
HBV or HCV	52	40	12
Negative	8	5	3
Liver function				1.000
Child-Pugh A	46	35	11
Child-Pugh B	14	10	4
AFP (μg/L)				0.125
≤ 400	35	29	6
> 400	23	15	8
Tumor number				0.174
Single	35	24	11
Multiple	25	21	4
Vascular invasion				0.122
Positive	22	19	3
Negative	38	26	12
Edmondson classification				0.019
I/II	21	12	9
III/IV	39	33	6

HBV: Hepatitis B virus; HCV: Hepatitis C virus; AFP: Alpha-fetal protein.

Table 6 Survival analysis of patients with different methylation status

Gene	M/U	n	Disease free survival				Overall survival
			Estimate (mo)	Scope (mo)	Log-Rank	P value	Estimate (mo)	Scope (mo)	Log-Rank	P value
SPARC	M	37	15.0	9.6-20.4	2.094	0.148	28.0	17.8-38.2	4.096	0.043
	U	14	24.0	12.6-35.5			41.0	36.5-45.5

SPARC: Secreted protein acidic and rich in cysteine; M: Methylation; U: Unmethylation.

Table 7 Cox regression model of overall survival

Factors	Univariate analysis			Multivariate analysis
	RR	95% CI	P value	RR	95% CI	P value
Methylation
Positive	2.672	0.999-7.147	0.044	3.207	1.290-7.975	0.012
Negative	1			1
Tumor size (cm)
> 5	5.293	1.560-17.959	0.008	8.045	2.125-30.456	0.002
≤ 5	1			1
AFP (μg/L)
> 400	3.306	1.421-7.694	0.006	7.105	1.798-28.080	0.005
≤ 400	1			1
Age (yr)
> 53	0.663	0.279-1.576	0.353
≤ 53	1
Gender
Male	1.104	0.373-3.266	0.859
Female	1
Tumor number
Multiple	3.330	1.440-7.704	0.005
Single	1
Vascular invasion
Positive	2.776	1.186-6.502	0.019
Negative	1
Edmondson classification
I/II	0.379	0.147-0.982	0.046
III/IV	1

AFP: Alpha-fetal protein; RR: Relative risk.