Gene expression in tumors and normal tissues
Presentation of STAB1 and STAB2 expression data from the TCGA dataset on a scatter plot showed that these two factors have different expressions levels (Fig. 1a, b). However, STAB1 and STAB2 were not differentially expressed (Fig. 1c, d) in tumor tissues and normal tissues of the GEO dataset. In addition, 7% of cancer samples showed STAB1 mutations, including missense mutation, truncating mutation, amplification, and deep deletion. Further, 5% of cancer samples showed mutations in the STAB2 gene including missense mutation, truncating mutation, amplification (Fig. 1e).
Basic patient information
Detailed information on the 370 HCC patients in TCGA database is shown in Table 1. BMI and TNM staging showed a significant correlation with MST (p = 0.021 and <0.001, respectively). Information on patients evaluated in the GEO database is shown in Table 2. Major tumor size, TNM stage, cirrhosis, BCLC stage, and alpha-fetoprotein levels were significantly correlated with OS (p = 0.003, 0.019, <0.001, <0.001, <0.001, 0.032, 0.019, and 0.026, respectively). Further, Gender, TNM stage, and BCLC stage were significantly correlated with RFS (p = 0.016, <0.001, and <0.001, respectively).
Table 1 Basic characteristics of HCC patients in the TCGA database
Variables
|
Patients
|
No. of events
|
MST
|
HR
|
Log-rank P
|
|
(n=370)
|
(%)
|
(months)
|
(95%CI)
|
|
Race
|
|
|
|
|
0.127
|
Asian
|
157
|
44(28.0%)
|
82
|
Ref
|
|
White+others
|
203
|
81(40.1%)
|
68
|
1.42(0.98-2.05)
|
|
Missing
|
10
|
|
|
|
|
Gender
|
|
|
|
|
0.488
|
Male
|
249
|
79(31.7%)
|
57
|
Ref
|
|
Female
|
121
|
51(42.1%)
|
59
|
1.07(0.89-1.28)
|
|
Age
|
|
|
|
|
0.248
|
≤60
|
177
|
55(31.1%)
|
71
|
Ref
|
|
>60
|
193
|
75(38.9%)
|
52
|
1.23(0.87-1.75)
|
|
BMI
|
|
|
|
|
0.021
|
≤25
|
177
|
61(34.5%)
|
68
|
Ref
|
|
>25
|
157
|
51(32.5%)
|
66
|
0.82(0.64-1.04)
|
|
Missing
|
36
|
|
|
|
|
TNM staging
|
|
|
|
|
<0.001
|
Ⅰ+Ⅱ
|
274
|
76(27.7%)
|
84
|
Ref
|
|
Ⅲ+Ⅳ
|
94
|
54(57.4%)
|
26
|
2.40(1.69-3.41)
|
|
Missing
|
2
|
|
|
|
|
Family history
|
|
|
|
|
0.192
|
Yes
|
112
|
49(44.1%)
|
40
|
Ref
|
|
No
|
207
|
69(33.3%)
|
62
|
0.78(0.54-1.13)
|
|
Missing
|
51
|
|
|
|
|
MST, median survival time; HR, hazard ratio; 95%CI, 95% confidence interval; Ref, reference; BMI, body mass index; TNM stage, tumor, node and metastasis stage.
Survival analysis
The TCGA data were analyzed using a multivariate Cox regression model with adjustment for race, gender, age, BMI, TNM stage, and family history, which showed no significant correlation between STAB1 and STAB2 with prognosis (adjusted p=0.300 and 0.865 respectively) (Fig. 2a, b, g).GEO data were evaluated by a multiple Cox regression model, with adjustment for race, age, HBV, ALT, major tumor size, multiple nodule status, liver cirrhosis, TNM staging, BCLC staging, and AFP, showing that STAB2 was significantly correlated with OS (HR = 0.541; 95% CI, 0.339–0.865; adjusted p = 0.010) and RFS (HR = 0.554; 95% CI, 0.376-0.816; adjusted p = 0.003) after adjustment (Fig. 2c, d, e, f, h, i).
Stratified chi-square test
We performed a hierarchical chi-square test on all clinical features. The results showed that high expression of STAB2 implied a more favorable prognosis in male, HBV (CC + NO), ALT> 50u / L, non-multinodular, cirrhosis, early TNM(I and II), early BCLC (0 and A) and AFP<=300ng/ml (p=0.013, 0.008, 0.039, 0.040,0.024,0.018,0.010 and 0.038 respectively). (Table 3)
Functional enrichment analysis
GO analysis showed STAB2 gene assembly in Hyaluronan catabolic process, hyaluronan metabolic process, regulation of calcineurin−NFAT signaling cascade, aminoglycan catabolic process, glycosaminoglycan catabolic process, mucopolysaccharide metabolic process, aminoglycan metabolic process, glycosaminoglycan metabolic process, carbohydrate derivative catabolic process, glycosaminoglycan binding, organonitrogen compound catabolic process cellular response to fibroblast growth factor stimulus, regulation of cell−substrate adhesion, regulation of apoptotic signaling pathway and cell migration. KEGG pathway enrichment showed that members of the STAB gene family is implicated in glycosaminoglycan degradation、vitamin digestion and absorption、fat digestion and absorption, ECM−receptor interaction、shigellosis、bile secretion、gastric acid secretion、cardiac muscle contraction, hematopoietic cell lineage、salivary secretion、pancreatic secretion、proteoglycans in cancer、thyroid hormone signaling pathway、adrenergic signaling in cardiomyocytes、cAMP signaling pathway、Epstein−Barr virus infection、and regulation of actin cytoskeleton. Details of the enrichment analysis are shown in Fig. 3b and Fig. 3b.
Analysis of the protein-protein interaction network using the STRING database shows that STAB1 and STAB2 are directly or intermediately related to some hub genes in HCC (such as CD44 and APOB) (Fig. 4 ).
Table 2. Basic characteristics of HCC patients in the GEO database
Variables
|
Patients
|
MST
|
OS
|
Log-rank P
|
MST
|
RFS
|
Log-rank P
|
(n=221)
|
HR(95%CI)
|
HR(95%CI)
|
Gender
|
|
|
|
0.149
|
|
|
0.016
|
Female
|
30
|
55
|
Ref
|
|
51
|
Ref
|
|
Male
|
191
|
48
|
1.70(0.82-3.52)
|
|
38
|
2.17(1.13-4.14)
|
|
Age
|
|
|
|
|
|
|
|
≤60
|
181
|
49
|
Ref
|
0.903
|
46
|
Ref
|
0.985
|
>60
|
40
|
48
|
0.97(0.55-1.69)
|
|
37
|
1.00(0.63-1.60)
|
|
HBV status
|
|
|
|
0.449
|
|
|
0.411
|
AVR-CC
|
56
|
45
|
Ref
|
|
29
|
Ref
|
|
CC+NO
|
162
|
50
|
0.91(0.12-6.74)
|
|
48
|
1.22(0.61-2.44)
|
|
Missing
|
3
|
|
|
|
|
|
|
ALT
|
|
|
|
0.726
|
|
|
0.229
|
≤50u/L
|
130
|
49
|
Ref
|
|
53
|
Ref
|
|
>50u/L
|
91
|
49
|
1.08(0.70-1.66)
|
|
40
|
1.25(0.87-1.78)
|
|
Main tumor size
|
|
|
|
0.003
|
|
|
0.100
|
≤5cm
|
140
|
53
|
Ref
|
|
51
|
Ref
|
|
>5cm
|
81
|
41
|
1.88(1.22-2.89)
|
|
30
|
1.36(0.94-1.97)
|
|
Multinodular
|
|
|
|
0.055
|
|
|
0.427
|
Yes
|
45
|
37
|
Ref
|
|
37
|
Ref
|
|
No
|
176
|
47
|
0.63(0.39-1.01)
|
|
41
|
0.84(0.54-1.30)
|
|
Cirrhosis
|
|
|
|
0.019
|
|
|
0.056
|
Yes
|
203
|
48
|
Ref
|
|
39
|
Ref
|
|
No
|
18
|
64
|
0.22(0.05-0.88)
|
|
54
|
0.46(0.20-1.04)
|
|
TNM staging
|
|
|
|
<0.001
|
|
|
<0.001
|
Ⅰ+Ⅱ
|
169
|
54
|
Ref
|
|
53
|
Ref
|
|
Ⅲ
|
49
|
30
|
0.65(0.33-1.30)
|
|
18
|
0.79(0.19-3.19)
|
|
Missing
|
3
|
|
|
|
|
|
|
BCLC staging
|
|
|
|
<0.001
|
|
|
<0.001
|
0+A
|
168
|
54
|
Ref
|
|
45
|
Ref
|
|
B+C
|
51
|
30
|
0.52(0.26-1.05)
|
|
33
|
0.69(0.35-1.35)
|
|
Missing
|
2
|
|
|
|
|
|
|
AFP
|
|
|
|
0.032
|
|
|
0.229
|
≤300ng/ml
|
118
|
53
|
Ref
|
|
49
|
Ref
|
|
>300ng/ml
|
100
|
44
|
1.63(1.06-2.50)
|
|
31
|
1.24(0.87-1.78)
|
|
Missing
|
3
|
|
|
|
|
|
|
OS, overall survival; RFS, recurrence-free survival; HBV status , hepatitis B virus status ; AVR–CC, active viral replication chronic carrier; CC, chronic carrier; ALT, alanine aminotransferase; AFP, alpha fetoprotein; TNM stage, tumor, node and metastasis stage ; BCLC staging, Barcelona Clinic Liver Cancer.
Table 3. Stratified chi-square test of basic characteristics of HCC patients in the GEO database
Variables
|
Patients
|
No. of events
|
MST
|
OS
|
Log-rank P
|
(n=221)
|
(%)
|
(months)
|
HR(95%CI)
|
Gender
|
|
|
|
|
|
Female
|
|
|
|
|
0.734
|
low expression
|
13
|
3(23.1%)
|
56
|
Ref
|
|
high expression
|
17
|
5(29.4%)
|
54
|
1.28(0.31-5.37)
|
|
Male
|
|
|
|
|
0.013
|
low expression
|
97
|
48(49.5%)
|
44
|
Ref
|
|
high expression
|
94
|
29(30.9%)
|
52
|
0.56(0.35-0.89)
|
|
HBV status
|
|
|
|
|
|
AVR-CC
|
|
|
|
|
0.865
|
low expression
|
31
|
14(45.2%)
|
46
|
Ref
|
|
high expression
|
25
|
11(44.0%)
|
45
|
1.07(0.49-2.36)
|
|
CC+NO
|
|
|
|
|
0.008
|
low expression
|
76
|
36(47.4%)
|
45
|
Ref
|
|
high expression
|
86
|
23(26.7%)
|
55
|
0.50(0.29-0.84)
|
|
Missing
|
3
|
|
|
|
|
ALT
|
|
|
|
|
|
≤50u/L
|
|
|
|
|
0.191
|
low expression
|
63
|
27(42.9%)
|
46
|
Ref
|
|
high expression
|
67
|
21(31.3%)
|
51
|
0.68(0.39-1.21)
|
|
>50u/L
|
|
|
|
|
0.039
|
low expression
|
47
|
24(51.1%)
|
43
|
Ref
|
|
high expression
|
44
|
13(29.5%)
|
54
|
0.49(0.25-0.96)
|
|
Multinodular
|
|
|
|
|
|
Yes
|
|
|
|
|
0.413
|
low expression
|
26
|
15(57.7%)
|
41
|
Ref
|
|
high expression
|
19
|
8(42.1%)
|
47
|
0.70(0.30-1.65)
|
|
No
|
|
|
|
|
0.04
|
low expression
|
84
|
36(42.9%)
|
46
|
Ref
|
|
high expression
|
92
|
26(28.3%)
|
54
|
0.59(0.36-0.98)
|
|
Cirrhosis
|
|
|
|
|
|
Yes
|
|
|
|
|
0.024
|
low expression
|
102
|
50(49.0%)
|
44
|
Ref
|
|
high expression
|
101
|
33(32.7%)
|
51
|
0.60(0.39-0.94)
|
|
No
|
18
|
|
|
|
0.892
|
low expression
|
|
1(12.5%)
|
56
|
Ref
|
|
high expression
|
10
|
1(10.0%)
|
63
|
0.83(0.05-13.21)
|
|
TNM staging
|
|
|
|
|
|
Ⅰ+Ⅱ
|
|
|
|
|
0.018
|
low expression
|
83
|
33(39.8%)
|
50
|
Ref
|
|
high expression
|
86
|
20(23.3%)
|
58
|
0.51(0.29-0.89)
|
|
Ⅲ
|
|
|
|
|
0.422
|
low expression
|
24
|
17(70.8%)
|
28
|
Ref
|
|
high expression
|
25
|
14(56.0%)
|
32
|
0.75(0.37-1.52)
|
|
Missing
|
3
|
|
|
|
|
BCLC staging
|
|
|
|
|
|
0+A
|
|
|
|
|
0.01
|
low expression
|
83
|
33(39.8%)
|
50
|
Ref
|
|
high expression
|
85
|
18(21.2%)
|
58
|
0.47(0.26-0.83)
|
|
B+C
|
|
|
|
|
0.756
|
low expression
|
25
|
17(68.0%)
|
28
|
Ref
|
|
high expression
|
26
|
16(61.5%)
|
31
|
0.90(0.45-1.78)
|
|
Missing
|
2
|
|
|
|
|
AFP
|
|
|
|
|
|
≤300ng/ml
|
|
|
|
|
0.038
|
low expression
|
49
|
22(44.9%)
|
49
|
Ref
|
|
high expression
|
69
|
17(24.6%)
|
56
|
0.51(0.27-0.96)
|
|
>300ng/ml
|
|
|
|
|
0.378
|
low expression
|
59
|
29(49.2%)
|
41
|
Ref
|
|
high expression
|
41
|
17(41.5%)
|
47
|
0.76(0.42-1.39)
|
|
Missing
|
3
|
|
|
|
|
OS, overall survival; RFS, recurrence-free survival; HBV status , hepatitis B virus status ; AVR–CC, active viral replication chronic carrier; CC, chronic carrier; ALT, alanine aminotransferase; AFP, alpha fetoprotein; TNM stage, tumor, node and metastasis stage ; BCLC staging, Barcelona Clinic Liver Cancer.