Retrospective Study
Copyright ©The Author(s) 2015.
World J Gastroenterol. Apr 7, 2015; 21(13): 3960-3969
Published online Apr 7, 2015. doi: 10.3748/wjg.v21.i13.3960
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
PrimerSequence (5'3')Amplicon (bp)
RT-PCR
IRAK3-ForwardATGCAGTGTAAGAAGCATTGGA247
IRAK3-ReverseGCAGGTAGTGAATGGCTTTGG
GLOXD1-ForwardCCCTTCCTACCCGGCTTCA122
GLOXD1-ReverseTGGAACCAGCGCAAAAGTGT
Pyrosequencing
GLOXD1-ForwardGAAGGGAGGTTTAGTGTTTAAGGA242
GLOXD1-ReverseAGCTGGACATCACCTCCCACAACGCCACCCCAACCAAAAACA
Universal primerAGCTGGACATCACCTCCCACAACG-Biotin
Sequencing primerAGGTTTAGTGTTTAAGGAT
MSP/Q-MSP
IRAK3-ForwardAGGAGATCGTTTAGTCGTGGGGTAC110
IRAK3-ReverseACCTCTACGATAAAAACGAAACTACCG
IRAK3-ProbeCTACCGAAACAAACAAAATA
GLOXD1-ForwardAGGATGTGATTAGGCGTGAGGTTC122
GLOXD1-ReverseAAAAAAACGAAACCCGTAACTCCG
GLOXD1-ProbeFAM-CGCTACTCTTTCCCC
Table 2 Clinicopathological characteristics of hepatocellular carcinoma patients
CharacteristicCases
Age, yr59 ± 14
Mean ± SD
Gender
Female94
Male66
Hepatitis
HBV-positive68
HCV-positive62
Double-negative30
Cirrhosis
No77
Yes80
Unknown3
Tumor size, cm
≤ 352
> 3108
Nodule
Solitary98
Multiple62
AFP level, ng/mL
≤ 1045
> 10113
Unknown2
Stage
I60
II46
III47
IV7
Invasion
No85
Yes75
Recurrence
No58
Yes36
Unknown66
Survival
No71
Yes27
Unknown62
Table 3 Methylation frequency of IRAK3 and GLOXD1 in liver tissues
SymbolM-Index1cut-off valueNo. of methylated cases/total
P value2
Normal controlsNontumor tissuesHCC tissues
IRAK38.901/29 (3.4%)23/160 (14.4%)102/160 (63.8%)< 0.0001
GLOXD10.602/29 (6.9%)4/160 (2.5%)75/160 (46.9%)< 0.0001
Table 4 Association between gene methylation and clinicopathological characteristics of 160 hepatocellular carcinoma patients
Methylation statusIRAK3, M-Index
P valueGLOXD1, M-Index
P value
Characteristic8.90> 8.900.60> 0.60
Cases581028575
Age, yr
≤ 5927471.0045290.08
> 5931554046
Gender
Female25410.7439270.26
Male33614648
Hepatitis
HBV-positive21470.3238300.22
HCV-positive23392834
Double-negative14161911
Cirrhosis
No33440.0746310.15
Yes23573842
Unknown2112
Tumor size, cm
≤ 316360.3825270.40
> 342666048
Nodule
Solitary37610.7453450.87
Multiple21413230
AFP level, ng/mL
≤ 1014310.5820250.16
> 1042716548
Unknown2002
Stage
I22380.0328320.66
II23232620
III13342720
IV0743
Invasion
No30550.8739460.06
Yes28474629
Recurrence
No22360.3834240.83
Yes10251916
Unknown26413235
Survival
No25460.4738330.26
Yes720189
Unknown26362933