1.1 SST methylation rate in GIT cancers
We detected the methylation rates of 15 CpG sites, which were located in +18, +25, +34, +42, +44, +85, +92, +94, +97, +100, +116, +127, +129, +138, +148.(Figure 1)
The SST methylation rates in EC, GC, and CC were shown in Table 1, Figure 2.
In 42 cases of EC, the average methylation rate in cancer tissue of EC was significantly higher than that in control tissue (p<0.05), including the +18,+34,+42,+44,+92,+94,+100,+116,+127,+129,+138 and +148 CpG sites (p <0.05).
In 99 cases of GC, the average methylation rate in cancer tissue was significantly higher than that in control tissue (p <0.05), including the +18, +42,+44, +94,+100, +127 and +129 CpG sites (all p <0.05).
In 70 cases of CC, the average methylation rate in cancer tissue was significantly
higher than that in control tissue (p <0.05), including the +18,+34,+42,+44, +92, +94, +97, +100, +116, +127, +129, +138 and +148 CpG sites (all p <0.05).
Joint analysis for the methylation difference of CpG sites in SST promoters for the three cancers showed that the methylation rates of CpG sites 7 sites including +18, +42, +44, +94, +100, +127, and +129 were meaningful significantly different from the adjacent control tissue. However, but the differences of CpG sites +25 and +85 were not statistically significant. The CpG sites of +34, +92 and +116 were significantly different in EC and CC, however, no significant difference was observed in GC. The CpG site +97 was significantly different in CC but not in EC and GC. The CpG sites +138 and +148 were significantly different in EC but not in the others.
Table 1 Methylation rates of CpG sites in GIT cancers
CpG site
|
Mean±Std
|
P Value
|
EC
|
EN
|
GC
|
GN
|
CRC
|
CRN
|
EC/EN
|
GC/GN
|
CRC/CRN
|
9
|
0.457±0.157
|
0.317±0.105
|
0.416±0.116
|
0.359±0.111
|
0.425±0.126
|
0.374±0.104
|
<0.001
|
<0.001
|
0.005
|
10
|
0.495±0.136
|
0.45±0.132
|
0.494±0.097
|
0.482±0.12
|
0.495±0.097
|
0.475±0.102
|
0.129
|
0.404
|
0.182
|
11
|
0.521±0.154
|
0.416±0.131
|
0.51±0.102
|
0.486±0.089
|
0.537±0.105
|
0.488±0.071
|
0.003
|
0.098
|
0.001
|
12
|
0.53±0.15
|
0.438±0.143
|
0.556±0.123
|
0.492±0.091
|
0.607±0.119
|
0.526±0.053
|
0.004
|
<0.001
|
<0.001
|
13
|
0.541±0.158
|
0.438±0.146
|
0.545±0.144
|
0.489±0.101
|
0.614±0.122
|
0.529±0.055
|
0.002
|
0.002
|
<0.001
|
14
|
0.483±0.121
|
0.469±0.117
|
0.479±0.122
|
0.474±0.091
|
0.518±0.101
|
0.52±0.073
|
0.565
|
0.734
|
0.9
|
15
|
0.72±0.098
|
0.668±0.117
|
0.678±0.11
|
0.669±0.085
|
0.74±0.099
|
0.708±0.067
|
0.032
|
0.551
|
0.019
|
16
|
0.664±0.125
|
0.57±0.136
|
0.62±0.132
|
0.576±0.099
|
0.698±0.116
|
0.62±0.081
|
0.002
|
0.009
|
<0.001
|
17
|
0.575±0.129
|
0.538±0.127
|
0.58±0.138
|
0.547±0.1
|
0.646±0.122
|
0.576±0.075
|
0.188
|
0.061
|
<0.001
|
18
|
0.624±0.111
|
0.568±0.121
|
0.602±0.132
|
0.568±0.106
|
0.691±0.111
|
0.623±0.067
|
0.017
|
0.041
|
<0.001
|
19
|
0.781±0.095
|
0.701±0.118
|
0.715±0.088
|
0.69±0.088
|
0.765±0.098
|
0.722±0.067
|
0.001
|
0.061
|
0.002
|
20
|
0.808±0.09
|
0.703±0.126
|
0.744±0.103
|
0.705±0.097
|
0.774±0.094
|
0.73±0.069
|
<0.001
|
0.009
|
0.001
|
21
|
0.827±0.091
|
0.708±0.131
|
0.765±0.104
|
0.711±0.105
|
0.799±0.095
|
0.753±0.063
|
<0.001
|
0.001
|
0.001
|
22
|
0.865±0.067
|
0.796±0.115
|
0.825±0.08
|
0.804±0.086
|
0.838±0.076
|
0.822±0.067
|
0.001
|
0.102
|
0.111
|
23
|
0.839±0.083
|
0.761±0.116
|
0.788±0.087
|
0.77±0.091
|
0.804±0.096
|
0.793±0.066
|
0.001
|
0.186
|
0.325
|
AVG
|
0.649±0.078
|
0.569±0.107
|
0.622±0.09
|
0.588±0.079
|
0.663±0.083
|
0.617±0.042
|
<0.001
|
0.008
|
<0.001
|
1.2 Correlation of methylation of SST and tumor biological behavior
We analyzed the methylation rate of SST promoter region in four tumor biological behaviors (Lymph node metastasis, Vascular tumor thrombus, Depth of infiltration and Differentiation). (Table 2)
In EC, two sites (+127, +129) were different in vascular tumor thrombus (0.861±0.071 vs. 0.794±0.090 p = 0.021, 0.878±0.068 vs. 0.813±0.093 p = 0.038). The methylation rates of the above two CpG sites in positive vascular cancer thrombus group was significantly higher than that in negative group. In the depth of infiltration, the muscular layer methylation rate at site +85 was the lowest, 0.422±0.105, serous layer was 0.507±0.117, and mucosa and submucosa were 0.534±0.172 (p = 0.041). At site +18, the methylation rate of poor differentiation was 0.616±0.137, and medium and high differentiation were 0.432±0.153 and 0.442±0.145 respectively (p = 0.014).
In GC, the methylation rate at site +25 was related to lymph node metastasis, and the positive group was significantly lower than the negative group (0.477±0.092 vs. 0.536±0.103 p = 0.013).
In CC, the methylation rate at position +94 was related to the depth of infiltration, and the serous layer was significantly higher than the muscular layer (0.718±0.098 vs. 0.649±0.129 p = 0.025). In this experiment, the infiltration depth of colorectal cancer did not have cases below the muscular layer.
1.3 SST mRNA expression in GIT cancers
The SST mRNA expressions in EC, GC, and CC were shown in Figure 3.
In 50 cases of EC, no statistically significant difference was found in SST mRNA expression between cancer and adjacent control tissue (0.0167 ± 0.0455 vs. 0.033 ± 0.1061, p = 0.32).
In 52 cases of GC, the expression of SST mRNA in cancer tissue was significantly lower than that in adjacent control tissue (0.0086 ± 0.0176 vs. 0.0318 ± 0.0404, p <0.001);
In 65 cases of CC, the expression of SST mRNA in cancer tissue was significantly lower than that in adjacent control tissue (0.0098 ± 0.0263 vs. 0.0819 ± 0.1372, p <0.001).
1.4 Correlation between SST promoter methylation and mRNA expression in GIT cancers
SST promoter methylation and SST mRNA expression had no significant correlation in EC group. In GC group, except CpG sites +25 and +85, the SST methylation and expression of other sites were negatively correlated (P <0.05). For CC group, except CpG sites of +25, +85 and +148, methylation rate was negatively correlated with expression (P <0.05). (Table 3)
Table 2 Correlation analysis of methylation of SST promoter region and tumor biological behavior of GIT cancers
Cancer
|
Parameter
|
Group
|
n
|
+18
|
+25
|
+34
|
+42
|
+44
|
+85
|
+92
|
+94
|
+97
|
+100
|
+116
|
+127
|
+129
|
+138
|
+148
|
AVG
|
EC
|
Lymphatic metastasis
|
Positive
|
25
|
0.497
|
0.654
|
0.71
|
0.311
|
0.405
|
0.858
|
0.617
|
0.749
|
0.798
|
0.265
|
0.405
|
0.828
|
0.969
|
0.497
|
0.788
|
0.513
|
|
|
Negetive
|
17
|
|
Vessel carcinoma embolus
|
Positive
|
9
|
0.718
|
0.976
|
0.786
|
0.587
|
0.414
|
0.249
|
0.526
|
0.651
|
0.303
|
0.171
|
0.303
|
0.021
|
0.038
|
0.075
|
0.098
|
0.224
|
|
|
Negetive
|
33
|
|
Depth of infiltration
|
serosa
|
26
|
0.141
|
0.451
|
0.92
|
0.201
|
0.268
|
0.041
|
0.478
|
0.668
|
0.099
|
0.035
|
0.094
|
0.4
|
0.51
|
0.316
|
0.355
|
0.641
|
|
|
muscular
|
13
|
|
|
mucosal and Submucosal
|
3
|
|
Differentiation
|
poorly
|
5
|
0.014
|
0.17
|
0.15
|
0.381
|
0.381
|
0.827
|
0.937
|
0.897
|
0.803
|
0.977
|
0.195
|
0.289
|
0.143
|
0.247
|
0.346
|
0.289
|
|
|
moderately
|
21
|
|
|
high
|
16
|
GC
|
Lymphatic metastasis
|
Positive
|
64
|
0.471
|
0.013
|
0.073
|
0.257
|
0.578
|
0.339
|
0.821
|
0.804
|
0.91
|
0.958
|
0.566
|
0.869
|
0.848
|
0.487
|
0.301
|
0.924
|
|
|
Negetive
|
27
|
|
Vessel carcinoma embolus
|
Positive
|
57
|
0.961
|
0.993
|
0.085
|
0.608
|
0.426
|
0.348
|
0.724
|
0.931
|
0.879
|
0.583
|
0.688
|
0.737
|
0.831
|
0.7
|
0.403
|
0.974
|
|
|
Negetive
|
34
|
|
Depth of infiltration
|
serosa
|
79
|
0.472
|
0.662
|
0.743
|
0.508
|
0.347
|
0.097
|
0.485
|
0.668
|
0.284
|
0.517
|
0.309
|
0.479
|
0.363
|
0.295
|
0.265
|
0.356
|
|
|
muscular
|
9
|
|
|
mucosal and Submucosal
|
3
|
|
Differentiation
|
poorly
|
75
|
0.608
|
0.658
|
0.811
|
0.725
|
0.667
|
0.119
|
0.775
|
0.991
|
0.551
|
0.884
|
0.446
|
0.499
|
0.462
|
0.349
|
0.364
|
0.685
|
|
|
moderately
|
12
|
|
|
high
|
4
|
CRC
|
Lymphatic metastasis
|
Positive
|
38
|
0.658
|
0.56
|
0.939
|
0.81
|
0.923
|
0.758
|
0.721
|
0.973
|
0.758
|
0.651
|
0.21
|
0.272
|
0.321
|
0.285
|
0.06
|
0.63
|
|
|
Negetive
|
42
|
|
Vessel carcinoma embolus
|
Positive
|
11
|
0.917
|
0.273
|
0.743
|
0.939
|
0.994
|
0.227
|
0.534
|
0.905
|
0.85
|
0.917
|
0.553
|
0.66
|
0.691
|
0.463
|
0.796
|
0.917
|
|
|
Negetive
|
69
|
|
Depth of infiltration
|
serosa
|
59
|
0.581
|
0.891
|
0.267
|
0.184
|
0.102
|
0.9
|
0.13
|
0.025
|
0.184
|
0.083
|
0.119
|
0.058
|
0.253
|
0.207
|
0.227
|
0.173
|
|
|
muscular
|
21
|
|
|
mucosal and Submucosal
|
0
|
|
Differentiation
|
poorly
|
25
|
0.76
|
0.36
|
0.53
|
0.289
|
0.151
|
0.519
|
0.24
|
0.081
|
0.351
|
0.271
|
0.426
|
0.139
|
0.497
|
0.647
|
0.334
|
0.157
|
|
|
moderately
|
40
|
|
|
high
|
15
|
Table 3 Correlation between SST promoter methylation and SST mRNA expression in GIT cancers
CpG site
|
r
|
P
|
EC
|
GC
|
CC
|
EC
|
GC
|
CC
|
+18
|
-0.063
|
-0.363
|
-0.23
|
0.57
|
< 0.001
|
0.008
|
+25
|
-0.13
|
-0.154
|
-0.17
|
0.239
|
0.118
|
0.053
|
+34
|
-0.071
|
-0.271
|
-0.23
|
0.518
|
0.005
|
0.008
|
+42
|
0.005
|
-0.393
|
-0.379
|
0.966
|
< 0.001
|
< 0.001
|
+44
|
0.026
|
-0.371
|
-0.396
|
0.816
|
< 0.001
|
< 0.001
|
+85
|
0.025
|
-0.107
|
0.083
|
0.818
|
0.28
|
0.346
|
+92
|
-0.001
|
-0.226
|
-0.193
|
0.989
|
0.021
|
0.028
|
+94
|
-0.003
|
-0.355
|
-0.406
|
0.975
|
< 0.001
|
< 0.001
|
+97
|
0.083
|
-0.252
|
-0.36
|
0.453
|
0.01
|
< 0.001
|
+100
|
0.048
|
-0.211
|
-0.361
|
0.663
|
0.031
|
< 0.001
|
+116
|
-0.089
|
-0.252
|
-0.273
|
0.419
|
0.01
|
0.002
|
+127
|
-0.034
|
-0.255
|
-0.272
|
0.758
|
0.009
|
0.002
|
+129
|
-0.047
|
-0.334
|
-0.306
|
0.668
|
0.001
|
<0.001
|
+138
|
0.074
|
-0.292
|
-0.179
|
0.501
|
0.003
|
0.041
|
+148
|
0.045
|
-0.212
|
-0.129
|
0.687
|
0.031
|
0.144
|
AVG
|
-0.026
|
-0.35
|
-0.374
|
0.813
|
< 0.001
|
< 0.001
|
1.5 The efficacy of SST promoter methylation for diagnosis of GIT cancers
Multivariate logistic regression was used to build appropriate diagnostic models. The diagnostic efficiency was assessed with sensitivity, specificity, Yorden Index and AUC of ROC. The combination of 8 CpG sites (+18, +42,+44,+85,+92,+94,+129,+138) had the largest area under the curve (AUC) of 0.817 with a sensitivity of 76.5% and a specificity of 75.0% GIT cancers. For GIT cancers, upper gastrointestinal tract, gastrointestinal, EC, GC, and CC models were established with AUC above 0.8. Co-differential sites that were significantly negatively correlated with expression in the first model were selected for modeling. In EC, the combination of 2 CpG sites (+18, +129) had the best diagnostic efficiency of AUC at 0.818 with a sensitivity of 80.0% and a specificity of 72.3%. (Table 4, Figure 4)
Table 4 Diagnostic value of combinations of CpG sites
CpG sites
|
Tumor
|
AUC
|
Sen
|
Spe
|
YD
|
Cut off
|
+18,+42,+44,+85,+92,
+94,+129,+138
|
Gastrointestinal tract tumor
|
0.817
|
0.765
|
0.75
|
0.515
|
50%
|
+18,+92,+94,+129,+138
|
Upper digestive tract tumor
|
0.816
|
0.681
|
0.849
|
0.53
|
57.20%
|
+42,+44,+85,+92,+94,
+116,+129,+138
|
Gastrointestinal tumor
|
0.819
|
0.685
|
0.865
|
0.55
|
58.10%
|
+18,+25,+97,+129
|
Esophageal cancer
|
0.868
|
0.778
|
0.83
|
0.608
|
50.20%
|
+18,+92,+129,+148
|
Gastric cancer
|
0.812
|
0.657
|
0.838
|
0.495
|
57.40%
|
+44, +92, +94
|
Colorectal cancer
|
0.813
|
0.756
|
0.779
|
0.535
|
50.10%
|
+18, +129
|
Gastrointestinal tract tumor
|
0.713
|
0.65
|
0.681
|
0.331
|
49.70%
|
+18, +129
|
Upper digestive tract tumor
|
0.723
|
0.667
|
0.671
|
0.338
|
48.10%
|
+42
|
Gastrointestinal tumor
|
0.712
|
0.492
|
0.892
|
0.384
|
56.60%
|
+18, +129
|
Esophageal cancer
|
0.818
|
0.8
|
0.723
|
0.523
|
39.90%
|
+42, +129
|
Gastric cancer
|
0.682
|
0.576
|
0.727
|
0.303
|
52.80%
|
+44, +94
|
Colorectal cancer
|
0.796
|
0.732
|
0.814
|
0.546
|
49.40%
|