Figure 1Percentage of methylation ratio (PMR) contains nine cancer-associated genes in colorectal cancer patients. Promoter methylation of nine tumor-association genes in tumor tissues and corresponding adjacent normal tissues from 100 patients with colorectal cancers and fourteen normal colorectal tissues was measured by means of quantitative methylation reverse transcription polymerase chain reaction. Nr, normal colorectal tissues; Adj, adjacent normal colorectal tissues; Tu, colorectal cancer tissues.
Table 1.Primer and probe sequences for quantitative methylation reverse transcription polymerase chain reaction
Genes |
Genebank |
Size (bp) |
Primer and probe sequences (5 ‘ → 3 ‘)
|
Forward |
Reverse |
APC |
U02509 |
73 |
GAACCAAAACGCTCCCCAT |
TTATATGTCGGTTACGTGCGTTTATAT |
|
|
|
(Probe; 6FAM-CCCGTCGAAAACCCGCCGATTA-MGB) |
p16INK4a
|
NM_000077 |
67 |
TGGAATTTTCGGTTGATTGGT |
AACAACGTCGCACCTCCT |
|
|
|
(Probe; 6FAM- ACCCGACCCCGAACCGCG-MGB) |
RASSF1A |
AC002481 |
64 |
ATTGAGTTGCGGGAGTTGGT |
ACACGCTCCAACCGAATACG |
|
|
|
(Probe; 6FAM- CCCTTCCCAACGCGCCCA-MGB) |
Twist1 |
NM_011658 |
70 |
GTAGCGCGGCGAACGT |
AAACGCAACGAATCATAACCAAC |
|
|
|
(Probe; 6FAM- CCAACGCACCCAATCGCTAAACGA-MGB) |
TIMP3 |
U33110 |
92 |
GCGTCGGAGGTTAAGGTTGTT |
CTCTCCAAAATTACCGTACGCG |
|
|
|
(Probe; 6FAM- AACTCGCTCGCCCG CCGAA-MGB) |
E-Cadherin |
L34545 |
69 |
AATTTTAGGTTAGAGGGTTATCGCGT |
TCCCCAAAACGAAACTAACGAC |
|
|
|
(Probe; 6FAM- CGCCCACCCGACCTCGCAT-MGB) |
ABCB1 |
NM_000927 |
75 |
TCGGGTCGGGAGTAGTTATTTG |
CGACTATACTCAACCCACGCC |
|
|
|
(Probe; 6FAM- ACGCTATTCCTACCCAACCAATCAACCTCA-MGB) |
COX2 |
AF044206 |
145 |
CGGAAGCGTTCGGGTAAAG |
AATTCCACCGCCCCAAAC |
|
|
|
(Probe; 6FAM- TTTCCGCCAAATATCTTTTCTTCTTCGCA-MGB) |
Smad4 |
NM_005359.5 |
142 |
GTAATAATACGGTTTTGGTCGTC |
CTCCCACCCCCTAAACGACCGCG |
|
|
|
(Probe; 6FAM-CGTTCGCGTTTTTTCGTT-MGB) |
ACTB |
Y00474 |
80 |
TCGAAATTTTCGTTGTTGCGT |
TATCCGTACCTACCGCCGC |
|
|
|
(Probe; 6FAM- ACCATACCCAACTTCGCCGACACCTAA-MGB) |
Table 2.Average PMR and hypermethylation frequency over PMR 10 according to gene and tissue type
Genes |
Tissues (n) |
Average PMR (%) |
PMR 10 frequency |
p-value
|
Nr vs. Adj |
Nr vs. Tu |
Adj vs. Tu |
APC |
Nr (14) |
5.0 |
2 (14.3) |
<0.01 |
<0.01 |
<0.01 |
|
Adj (100) |
13.5 |
38 (38.0) |
|
|
|
|
Tu (100) |
38.6 |
52 (52.0) |
|
|
|
p16INK4a
|
Nr (14) |
1.4 |
0 (0.0) |
0.02 |
<0.01 |
0.28 |
|
Adj (100) |
5.5 |
10 (10.0) |
|
|
|
|
Tu (100) |
8.8 |
14 (14.0) |
|
|
|
RASSF1A |
Nr (14) |
1.0 |
0 (0.0) |
<0.01 |
<0.01 |
<0.01 |
|
Adj (100) |
3.1 |
9 (9.0) |
|
|
|
|
Tu (100) |
7.0 |
20 (20.0) |
|
|
|
Twist1 |
Nr (14) |
3.0 |
1 (7.1) |
<0.01 |
<0.01 |
<0.01 |
|
Adj (100) |
22.0 |
41 (41.0) |
|
|
|
|
Tu (100) |
49.0 |
58 (58.0) |
|
|
|
TIMP3 |
Nr (14) |
12.2 |
9 (64.3) |
<0.01 |
<0.01 |
0.36 |
|
Adj (100) |
36.2 |
62 (62.0) |
|
|
|
|
Tu (100) |
44.5 |
43 (43.0) |
|
|
|
E-cadherin |
Nr (14) |
11.9 |
11 (78.6) |
0.11 |
0.16 |
0.49 |
|
Adj (100) |
7.5 |
29 (29.0) |
|
|
|
|
Tu (100) |
8.7 |
28 (28.0) |
|
|
|
ABCB1 |
Nr (14) |
14.5 |
8 (57.1) |
0.39 |
<0.01 |
<0.01 |
|
Adj (100) |
11.0 |
30 (30.0) |
|
|
|
|
Tu (100) |
60.1 |
69 (69.0) |
|
|
|
COX2 |
Nr (14) |
10.0 |
9 (64.3) |
0.04 |
<0.01 |
<0.01 |
|
Adj (100) |
19.0 |
27 (27.0) |
|
|
|
|
Tu (100) |
40.0 |
51 (51.0) |
|
|
|
Smad4 |
Nr (14) |
1.6 |
0 (0.0) |
<0.01 |
<0.01 |
<0.01 |
|
Adj (100) |
8.0 |
19 (19.0) |
|
|
|
|
Tu (100) |
21.0 |
39 (39.0) |
|
|
|
Table 3.Hypermethylation frequencies over PMR 10 of nine cancer-associated genes with clinical parameters in 100 colorectal cancer tissues
(n) |
APC
|
p16INK4a
|
RASSF1A
|
Twist1
|
TIMP3
|
E-canherin
|
ABCB1
|
COX2
|
Smad4
|
Freq |
p-value |
Freq |
p-value |
Freq |
p-value |
Freq |
p-value |
Freq |
p-value |
Freq |
p-value |
Freq |
p-value |
Freq |
p-value |
Freq |
p-value |
Age (y) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
<60 (48) |
21 (43.8) |
0.43 |
6 (12.5) |
0.22 |
6 (12.5) |
0.06 |
29 (60.4) |
0.81 |
23 (47.9) |
0.67 |
9 (18.8) |
0.1 |
36 (75.0) |
0.09 |
21 (43.8) |
0.69 |
17 (35.4) |
0.96 |
≥60 (52) |
31 (59.6) |
|
8 (15.4) |
|
14 (26.9) |
|
29 (55.8) |
|
20 (38.5) |
|
19 (36.5) |
|
33 (63.5) |
|
30 (57.7) |
|
22 (42.3) |
|
Gender |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Male (51) |
29 (56.9) |
0.08 |
6 (11.8) |
0.68 |
13 (25.5) |
0.19 |
29 (56.9) |
0.64 |
23 (45.1) |
0.34 |
18 (35.3) |
0.05 |
39 (76.5) |
0.08 |
27 (52.9) |
0.16 |
20 (39.2) |
0.48 |
Female (49) |
23 (46.9) |
|
8 (16.3) |
|
7 (14.3) |
|
29 (59.2) |
|
20 (40.8) |
|
10 (20.4) |
|
30 (61.2) |
|
24 (49.0) |
|
19 (38.8) |
|
Smoking |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Ever (20) |
12 (60.0) |
0.48 |
2 (10.0) |
0.291
|
3 (15.0) |
0.541
|
15 (75.0) |
0.09 |
6 (30.0) |
0.19 |
8 (40.0) |
0.18 |
15 (75.0) |
0.66 |
8 (40.0) |
0.27 |
7 (35.0) |
0.68 |
Never (80) |
40 (50.0) |
|
12 (15.0) |
|
17 (21.3) |
|
43 (53.8) |
|
37 (46.3) |
|
20 (25.0) |
|
54 (67.5) |
|
43 (53.8) |
|
32 (40.0) |
|
Alcohol |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Yes (21) |
12 (57.1) |
0.67 |
4 (19.0) |
1.001
|
4 (19.0) |
0.761
|
13 (61.9) |
0.68 |
7 (33.3) |
0.31 |
8 (38.1) |
0.25 |
17 (81.0) |
0.09 |
11 (52.4) |
0.89 |
9 (42.9) |
0.68 |
No (79) |
40 (50.6) |
|
10 (12.7) |
|
16 (20.3) |
|
45 (57.0) |
|
36 (45.6) |
|
20 (25.3) |
|
52 (65.8) |
|
40 (50.6) |
|
30 (38.0) |
|
Staging |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
I (3) |
2 (66.7) |
0.781
|
|
0.541
|
1 (33.3) |
0.711
|
1 (33.3) |
0.351
|
1 (33.3) |
0.721
|
2 (66.7) |
0.061
|
2 (66.7) |
0.30*
|
1 (33.3) |
0.211
|
1 (33.3) |
0.981
|
II (49) |
26 (53.1) |
|
8 (16.3) |
|
10 (20.4) |
|
31 (63.3) |
|
24 (49.0) |
|
18 (36.7) |
|
36 (73.5) |
|
30 (61.2) |
|
20 (40.8) |
|
III (40) |
21 (52.5) |
|
4 (10.0) |
|
8 (20.0) |
|
20 (50.0) |
|
15 (37.5) |
|
7 (17.5) |
|
27 (67.5) |
|
16 (40.0) |
|
15 (37.5) |
|
IV (8) |
3 (37.5) |
|
2 (25.0) |
|
1 (12.5) |
|
6 (75.0) |
|
3 (37.5) |
|
1 (12.5) |
|
4 (50.0) |
|
4 (50.0) |
|
3 (37.5) |
|
Table 4.Hypermethylation agreement (kappa values) among nine cancer-associated genes in 100 colorectal cancer tissues
Gene type |
APC |
p16INK4a
|
RASSF1A |
Twist1 |
TIMP3 |
E-cadherin |
ABCB1 |
COX2 |
p16INK4a
|
- 0.02 |
|
|
|
|
|
|
|
RASSF1A |
0.05 |
0.01 |
|
|
|
|
|
|
Twist1 |
0.17 |
- 0.01 |
0.02 |
|
|
|
|
|
TIMP3 |
0.25 |
0.22 |
0.02 |
0.16 |
|
|
|
|
E-cadherin |
0.32 |
0.06 |
0.511
|
0.18 |
0.21 |
|
|
|
ABCB1 |
0.14 |
0.00 |
0.09 |
0.38 |
0.24 |
0.27 |
|
|
COX2 |
0.20 |
0.11 |
- 0.05 |
0.26 |
0.32 |
0.19 |
0.36 |
|
Smad4 |
0.33 |
0.03 |
- 0.04 |
0.33 |
0.26 |
0.23 |
0.421
|
0.521
|