Methylation of IRAK3 is a novel prognostic marker in hepatocellular carcinoma

Table 1 Primer and probe sequences for reverse-transcription polymerase chain reaction, methylation-specific polymerase chain reaction, pyrosequencing, and quantitative methylation-specific polymerase chain reaction

Primer	Sequence (5'→3')	Amplicon (bp)
RT-PCR
IRAK3-Forward	ATGCAGTGTAAGAAGCATTGGA	247
IRAK3-Reverse	GCAGGTAGTGAATGGCTTTGG
GLOXD1-Forward	CCCTTCCTACCCGGCTTCA	122
GLOXD1-Reverse	TGGAACCAGCGCAAAAGTGT
Pyrosequencing
GLOXD1-Forward	GAAGGGAGGTTTAGTGTTTAAGGA	242
GLOXD1-Reverse	AGCTGGACATCACCTCCCACAACGCCACCCCAACCAAAAACA
Universal primer	AGCTGGACATCACCTCCCACAACG-Biotin
Sequencing primer	AGGTTTAGTGTTTAAGGAT
MSP/Q-MSP
IRAK3-Forward	AGGAGATCGTTTAGTCGTGGGGTAC	110
IRAK3-Reverse	ACCTCTACGATAAAAACGAAACTACCG
IRAK3-Probe	CTACCGAAACAAACAAAATA
GLOXD1-Forward	AGGATGTGATTAGGCGTGAGGTTC	122
GLOXD1-Reverse	AAAAAAACGAAACCCGTAACTCCG
GLOXD1-Probe	FAM-CGCTACTCTTTCCCC

Allele-specific primer sequences for MSP and Q-MSP are the same. RT-PCR: Reverse transcription-polymerase chain reaction; MSP: Methylation-specific polymerase chain reaction; Q-MSP: Quantitative MSP.

Table 2 Clinicopathological characteristics of hepatocellular carcinoma patients

Characteristic	Cases
Age, yr	59 ± 14
Mean ± SD
Gender
Female	94
Male	66
Hepatitis
HBV-positive	68
HCV-positive	62
Double-negative	30
Cirrhosis
No	77
Yes	80
Unknown	3
Tumor size, cm
≤ 3	52
> 3	108
Nodule
Solitary	98
Multiple	62
AFP level, ng/mL
≤ 10	45
> 10	113
Unknown	2
Stage
I	60
II	46
III	47
IV	7
Invasion
No	85
Yes	75
Recurrence
No	58
Yes	36
Unknown	66
Survival
No	71
Yes	27
Unknown	62

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

Table 3 Methylation frequency of IRAK3 and GLOXD1 in liver tissues

Symbol	M-Index1cut-off value	No. of methylated cases/total			P value2
		Normal controls	Nontumor tissues	HCC tissues
IRAK3	8.90	1/29 (3.4%)	23/160 (14.4%)	102/160 (63.8%)	< 0.0001
GLOXD1	0.60	2/29 (6.9%)	4/160 (2.5%)	75/160 (46.9%)	< 0.0001

¹Methylation-Index: the best cut-off value to discriminate 29 normal controls and 160 hepatocellular carcinoma (HCC) tissues;

²χ² test for trend.

Table 4 Association between gene methylation and clinicopathological characteristics of 160 hepatocellular carcinoma patients

Methylation status	IRAK3, M-Index		P value	GLOXD1, M-Index		P value
Characteristic	≤8.90	> 8.90		≤0.60	> 0.60
Cases	58	102		85	75
Age, yr
≤ 59	27	47	1.00	45	29	0.08
> 59	31	55		40	46
Gender
Female	25	41	0.74	39	27	0.26
Male	33	61		46	48
Hepatitis
HBV-positive	21	47	0.32	38	30	0.22
HCV-positive	23	39		28	34
Double-negative	14	16		19	11
Cirrhosis
No	33	44	0.07	46	31	0.15
Yes	23	57		38	42
Unknown	2	1		1	2
Tumor size, cm
≤ 3	16	36	0.38	25	27	0.40
> 3	42	66		60	48
Nodule
Solitary	37	61	0.74	53	45	0.87
Multiple	21	41		32	30
AFP level, ng/mL
≤ 10	14	31	0.58	20	25	0.16
> 10	42	71		65	48
Unknown	2	0		0	2
Stage
I	22	38	0.03	28	32	0.66
II	23	23		26	20
III	13	34		27	20
IV	0	7		4	3
Invasion
No	30	55	0.87	39	46	0.06
Yes	28	47		46	29
Recurrence
No	22	36	0.38	34	24	0.83
Yes	10	25		19	16
Unknown	26	41		32	35
Survival
No	25	46	0.47	38	33	0.26
Yes	7	20		18	9
Unknown	26	36		29	33

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