Trefoil factors: Tumor progression markers and mitogens via EGFR/MAPK activation in cholangiocarcinoma

Table 1 Cholangiocarcinoma samples tested for serum anti Opisthorchis viverrini antibody

No.	Code	Anti-OV titer (OD)1	OV positive
1	M009	0.804	+
2	M012	0.303	+
3	M030	0.820	+
4	M043	0.182	-
5	M055	0.185	-
6	M068	0.356	+
7	M071	0.257	+
8	M097	0.188	-
9	M101	0.235	+
10	M111	0.088	-
11	M114	0.285	+
12	M117	0.345	+
13	M119	0.168	-
14	M132	0.095	-
15	M137	0.330	+
16	M139	0.239	+
17	M142	0.344	+
18	M148	0.305	+
19	M152	0.108	-
20	M155	0.642	+
21	M157	0.482	+
22	M208	0.952	+
23	M214	0.188	-
24	M229	0.165	-
25	M240	0.669	+
26	M265	0.314	+
27	M279	0.502	+
28	N004	0.218	+
29	N021	0.232	+

¹Cut-off optical density (OD) = 0.200. OV: O. viverrini.

Table 2 Primer sequences of genomic and reverse transcription polymerase chain reaction, and polymerase chain reaction fragment sizes

Primer name	Forward (5→3)	Reverse (5→3)	Product size (bp)
Genomic-PCR
TFF1	CAGGGATCTGCCTGCATC	ATCGATCTCTTTTAATTTTTAGGCC	219
TFF2	GAAGAATCTCCCGAACCAGG	GTCACACTTCAAAAACTAGAGG	123
TFF3	CAGGCACTGTTCATCTCAGC	TATTCGTTAAGACATCAGGCTCC	129
β-actin	TCACCCACACTGTGCCCATCTACGA	CAGCGGAACCGCTCATTGCCAATGG	375
RT-PCR
TFF1	CCCGTGAAAGACAGAATT	GATCCCTGCAGAAGTGTCT	169
TFF2	CTCCTGGCAGCGCTCCTCGTC	GATGCCCGGGTAGCCACAGTTTCT	223
TFF3	AACCGGGGCTGCTGCTTTG	GAGGTGCCTCAGAAGGTGC	92
RPLP0	CTTCCCACTTGCTGAAAAG	CCAAATCCCATATCCTCGT	168

PCR: Polymerase chain reaction; RT-PCR: Reverse transcription PCR; TFF: Trefoil factor.

Table 3 Associations of trefoil factor protein expression in cholangiocarcinoma

TFF protein expression	n	TFF2			TFF3
		Negative	Positive	P-value	Negative	Positive	P-value
TFF1	110
Negative	54	42	12	NS	34	20	0.014
Positive	56	40	16		22	34
TFF2	110
Negative	82				44	38	NS
Positive	28				12	16

TFF: Trefoil factor.

Table 4 Relative copy number of trefoil factor genes in cholangiocarcinoma

No.	TFF1	TFF2	TFF3
1	2.031	1.691	2.311
2	1.431	1.371	1.721
3	1.551	1.371	2.341
4	2.001	2.191	2.911
5	1.461	1.731	1.841
6	1.791	1.781	3.211
7	1.821	2.741	3.671
8	1.441	0.96	1.381
9	1.551	0.98	1.781
10	1.551	0.99	1.561
11	1.681	1.30	2.361
12	1.681	1.10	1.401
13	1.971	0.85	2.171
14	1.611	1.33	2.321
15	1.581	1.00	1.581
16	1.621	1.22	2.891
17	1.491	1.17	1.351
18	1.521	0.97	2.251
19	1.621	1.21	1.851
20	1.14	1.10	1.551
21	1.17	0.98	1.661
22	0.83	0.92	1.571
23	1.14	1.03	1.481
24	0.98	1.14	1.411
25	1.06	1.15	1.651
26	1.06	1.33	2.131
27	1.32	0.91	1.421
28	1.20	1.01	1.571
29	1.27	1.631	1.851
30	1.32	1.891	1.581
31	1.29	2.681	2.161
32	1.28	1.381	1.871
33	0.88	1.481	1.631
34	1.491	1.421	1.24
35	1.381	0.71	0.99
36	1.771	1.03	1.12
37	1.381	0.92	1.29
38	1.511	1.17	0.94
39	1.521	1.02	1.23
40	1.461	1.12	1.34
41	1.421	0.97	1.34
42	1.371	0.73	0.79
43	0.84	0.472	0.58
44	0.63	0.372	0.88
45	0.68	0.532	0.86
46	0.63	0.512	0.83
47	0.86	0.382	0.55
48	0.76	0.422	0.462
49	0.92	0.68	0.452
50	0.82	0.85	0.512
51	1.07	0.79	0.97
52	1.15	0.59	1.11
53	0.95	0.84	1.02
54	0.84	0.79	0.68
55	0.74	0.77	1.26
56	0.77	1.03	0.83
57	1.13	1.31	1.26
58	1.13	1.03	1.20
59	1.00	0.88	0.89
60	1.17	0.70	1.15
61	0.94	0.91	1.19
62	0.76	0.72	0.63
63	1.15	1.04	1.15
64	1.25	0.95	0.87
65	1.01	0.77	0.90
66	1.11	1.27	1.03
67	0.85	0.62	0.54
68	1.10	0.96	1.23
69	1.06	0.92	0.84
70	1.05	0.88	0.80
71	1.12	1.09	1.10
72	1.33	0.87	1.22
73	0.87	1.01	0.71
74	0.75	0.88	0.76
75	1.04	0.74	1.14
76	1.10	0.67	0.93
77	1.06	0.98	1.14
78	1.10	0.97	1.30
79	1.22	0.87	1.21
80	1.16	0.95	1.32
81	1.13	1.07	1.32
82	1.10	0.88	1.24
83	0.92	1.05	1.31
84	1.14	0.97	1.09
85	1.01	0.98	1.14
86	1.13	1.07	1.26
87	0.91	0.59	1.23
88	0.97	0.69	1.16
89	0.91	0.78	0.91
90	1.06	0.71	1.06
91	1.15	0.69	0.88
92	0.96	0.58	0.57
93	1.03	1.00	1.30
94	1.20	0.93	1.06
95	1.25	0.76	0.77
96	1.28	1.21	1.24
97	1.28	1.15	0.94
98	1.10	0.82	0.99
99	1.28	0.73	0.71
100	0.87	0.59	0.91
101	1.05	0.78	1.28
102	1.00	0.70	0.99
103	0.66	0.61	0.61
104	0.89	0.74	0.80
105	0.88	0.57	0.60
106	0.82	0.56	0.58
107	1.04	1.05	1.04
108	0.90	0.98	0.88
109	0.65	0.65	0.64
110	1.18	1.21	1.20

Figures indicate relative copy number of trefoil factor (TFF) genes (TFF1, TFF2, TFF3) compared to the reference gene, β-actin.

¹Amplification;

²Deletion. Other figures are normal.