Clinical features of LIHC patients
Clinical as well as gene expression data for 374 primary cancer and 50 normal samples were downloaded from the TCGA database. They included patient age, histologicalal grade, pathologic stage, vascular invasion, survival status, gender, and T classification (Table 1).
Table 1
Clinical features of LIHC patients
Characteristic
|
levels
|
Overall
|
n
|
|
374
|
Age, n (%)
|
≤ 60
|
177 (47.5%)
|
|
> 60
|
196 (52.5%)
|
Gender, n (%)
|
Female
|
121 (32.4%)
|
|
Male
|
253 (67.6%)
|
histological grade, n (%)
|
G1
|
55 (14.9%)
|
|
G2
|
178 (48.2%)
|
|
G3
|
124 (33.6%)
|
|
G4
|
12 (3.3%)
|
Pathologic stage, n (%)
|
Stage I
|
173 (49.4%)
|
|
Stage II
|
87 (24.9%)
|
|
Stage III
|
85 (24.3%)
|
|
Stage IV
|
5 (1.4%)
|
Vascular invasion, n (%)
|
No
|
208 (65.4%)
|
|
Yes
|
110 (34.6%)
|
OS event, n (%)
|
Death
|
244 (65.2%)
|
|
Survival
|
130 (34.8%)
|
T stage, n (%)
|
T1
|
183 (49.3%)
|
|
T2
|
95 (25.6%)
|
|
T3
|
80 (21.6%)
|
|
T4
|
13 (3.5%)
|
Elevated AC008622.2 levels in LIHC patients
AC008622.2 transcript levels were analyzed in data from the TCGA database. AC008622.2 mRNA expressions were markedly elevated in LIHC tissues, relative to normal tissues (paired samples, P < 0.001; unpaired samples, P < 0.001). Patients at more advanced histologicalal stages had high AC008622.2 mRNA levels, relative to patients at less advanced histologicalal stages (P < 0.05). High-grade groups (G3/G4) exhibited high AC008622.2 mRNA levels than the low grade groups (G1/G2) (P < 0.001) (Figs. 1 and 2).
Diagnostic significance of AC008622.2 mRNA levels in LIHC
The ROC curve analysis was used to investigate the diagnostic significance of AC008622.2 mRNA levels in LIHC. Area under curve (AUC) for AC008622.2 mRNA expression levels was 0.958. With regards to diagnostic value at varying stages, the findings revealed a comparable diagnostic significance, with AUC values of 0.781, 0.746, 0.723 and 0.952 for stages I, II, III and IV, respectively (Fig. 3).
Association between clinical features and AC008622.2 mRNA levels in LIHC
Based on median AC008622.2 mRNA levels, samples were assigned into high and low groups. Elevated AC008622.2 mRNA levels correlated with histologicalal stage (P < 0.001) (Table 2).
Table 2
Relationship between AC008622.2 mRNA levels and clinical features in liver cancer
Characteristic
|
Low expression of AC008622.2
|
High expression of AC008622.2
|
p
|
Age, n (%)
|
|
|
0.277
|
<=60
|
83 (22.3%)
|
94 (25.2%)
|
|
> 60
|
104 (27.9%)
|
92 (24.7%)
|
|
Gender, n (%)
|
|
|
0.377
|
Female
|
56 (15%)
|
65 (17.4%)
|
|
Male
|
131 (35%)
|
122 (32.6%)
|
|
histological grade, n (%)
|
|
|
< 0.001
|
G1
|
37 (10%)
|
18 (4.9%)
|
|
G2
|
95 (25.7%)
|
83 (22.5%)
|
|
G3
|
48 (13%)
|
76 (20.6%)
|
|
G4
|
3 (0.8%)
|
9 (2.4%)
|
|
Pathologic stage, n (%)
|
|
|
0.065
|
Stage I
|
98 (28%)
|
75 (21.4%)
|
|
Stage II
|
38 (10.9%)
|
49 (14%)
|
|
Stage III
|
38 (10.9%)
|
47 (13.4%)
|
|
Stage IV
|
1 (0.3%)
|
4 (1.1%)
|
|
Vascular invasion, n (%)
|
|
|
0.063
|
No
|
115 (36.2%)
|
93 (29.2%)
|
|
Yes
|
48 (15.1%)
|
62 (19.5%)
|
|
T stage, n (%)
|
|
|
0.131
|
T1
|
102 (27.5%)
|
81 (21.8%)
|
|
T2
|
40 (10.8%)
|
55 (14.8%)
|
|
T3
|
36 (9.7%)
|
44 (11.9%)
|
|
T4
|
6 (1.6%)
|
7 (1.9%)
|
|
OS event, n (%)
|
|
|
0.002
|
Alive
|
137 (36.6%)
|
107 (28.6%)
|
|
Dead
|
50 (13.4%)
|
80 (21.4%)
|
|
High AC008622.2 mRNA levels is an independent risk factor for OS outcomes in LIHC patients
Kaplan–Meier analysis showed that elevated AC008622.2 mRNA levels were associated with poor OS (P < 0.001), shorter progress free interval (P < 0.001) and poor disease specific survival (P < 0.05) (Figs. 4 and 5). Subgroup analysis revealed that AC008622.2 mRNA levels markedly affected OS outcomes in LIHC cases of G1/G3 (P = 0.001), M0 (P < 0.001), T1/T2 (P = 0.001), T1/T3 (P < 0.001), clinical stage I/III (P < 0.001), and II/III (P < 0.001) (Fig. 5).
Univariate analysis showed that elevated AC008622.2 mRNA levels, T classification, and advanced stage correlated with OS outcomes. Multivariate analysis showed that AC008622.2 mRNA levels are independent risk factors for OS outcomes in LIHC (Table 3).
Table 3
Association between mRNA expressions of AC008622.2 and overall survival
Characteristics
|
Total(N)
|
Univariate analysis
|
Multivariate analysis
|
Hazard ratio (95% CI)
|
P value
|
Hazard ratio (95% CI)
|
P value
|
Gender
|
373
|
|
|
|
|
Male
|
252
|
Reference
|
|
|
|
Female
|
121
|
1.261 (0.885–1.796)
|
0.200
|
|
|
Age
|
373
|
|
|
|
|
<=60
|
177
|
Reference
|
|
|
|
> 60
|
196
|
1.205 (0.850–1.708)
|
0.295
|
|
|
T stage
|
370
|
|
|
|
|
T1
|
183
|
Reference
|
|
|
|
T2
|
94
|
1.431 (0.902–2.268)
|
0.128
|
0.000 (0.000-Inf)
|
0.994
|
T3
|
80
|
2.674 (1.761–4.060)
|
< 0.001
|
0.865 (0.118–6.326)
|
0.887
|
T4
|
13
|
5.386 (2.690-10.784)
|
< 0.001
|
1.740 (0.199–15.200)
|
0.616
|
Pathologic stage
|
349
|
|
|
|
|
Stage I
|
173
|
Reference
|
|
|
|
Stage II
|
86
|
1.417 (0.868–2.312)
|
0.164
|
4267218.741 (0.000-Inf)
|
0.994
|
Stage III
|
85
|
2.734 (1.792–4.172)
|
< 0.001
|
2.964 (0.406–21.644)
|
0.284
|
Stage IV
|
5
|
5.597 (1.726–18.148)
|
0.004
|
3.815 (0.340-42.846)
|
0.278
|
histological grade
|
368
|
|
|
|
|
G1
|
55
|
Reference
|
|
|
|
G2
|
178
|
1.162 (0.686–1.969)
|
0.576
|
|
|
G3
|
123
|
1.185 (0.683–2.057)
|
0.545
|
|
|
G4
|
12
|
1.681 (0.621–4.549)
|
0.307
|
|
|
Vascular invasion
|
317
|
|
|
|
|
No
|
208
|
Reference
|
|
|
|
Yes
|
109
|
1.344 (0.887–2.035)
|
0.163
|
|
|
Fibrosis ishak score
|
214
|
|
|
|
|
0
|
75
|
Reference
|
|
|
|
1/2
|
31
|
0.935 (0.437–2.002)
|
0.864
|
|
|
3/4
|
28
|
0.698 (0.288–1.695)
|
0.428
|
|
|
5/6
|
80
|
0.737 (0.410–1.325)
|
0.308
|
|
|
Adjacent hepatic tissue inflammation
|
236
|
|
|
|
|
None
|
118
|
Reference
|
|
|
|
Mild
|
101
|
1.204 (0.723–2.007)
|
0.476
|
|
|
Severe
|
17
|
1.144 (0.447–2.930)
|
0.779
|
|
|
AC008622 2
|
373
|
4.559 (2.065–10.068)
|
< 0.001
|
4.274 (1.885–9.689)
|
< 0.001
|
Relationships Between AC008622.2 levels and Immune Markers
Tumor infiltration is associated with LIHC prognosis[10]. Therefore, we tested whether the transcription levels of AC008622.2 in LIHC were correlated with immune infiltration. ESTIMATE can reveal infiltration level of immune cells by conducting single-sample GSEA (ssGSEA) according to the established immune signature, and low p-values correlate with high total infiltrations. These biomarkers were used for immune cell characterization, including B cells, T helper cells, macrophages, CD8 T cells, cytotoxic cells, NK cells, neutrophils and DCs in LIHC. The results showed that AC008622.2 was very weakly negatively correlated with counts of B cells, cytotoxic cells, NK cells, neutrophils, CD8 T cells, and DCs. AC008622.2 expressions were weakly associated with immune infiltrations of B cells and macrophages (Fig. 6).
Target Recognition Results
We obtained 46 potential target genes from the TCM database by analyzing 96 active ingredients in Danzhi Xiaoyao Powder, and all the target names were corrected to the gene names of the targets. A total of 1525 differentially expressed genes (DEGs) related to AC008622.2 were analysed using the DESeq2 program. These potential target genes and 1525 differentially expressed genes intersected, and 9 potential LIHC-related genes were identified by the intersection of a Venn diagram. Cytoscape software was used to develop a visualization network, which helped to understand the relationship between lncRNAs, potential targets and Danzhi Xiaoyao Powder (Fig. 7).