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©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Jun 7, 2009; 15(21): 2595-2601
Published online Jun 7, 2009. doi: 10.3748/wjg.15.2595
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
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 < 0.05P < 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