3.1 ISG20L2 is highly expressed in LUAD and High ISG20L2 expression is associated with poor LUAD prognosis
To investigate the role of ISG20L2 in LUAD, we initially analyzed its differential expression in LUAD mRNA using The Cancer Genome Atlas (TCGA) database[22] (https://genome-cancer.ucsc.edu/). Our analysis revealed that ISG20L2 expression was significantly upregulated in LUAD compared to corresponding healthy tissues (Fig. 1A; P<0.001). To confirm this finding, we performed immunohistochemical (IHC) staining on a chip containing 95 LUAD and 80 paracancerous specimens. The results demonstrated that ISG20L2 protein was highly expressed in lung adenocarcinoma tissues but exhibited lower expression in paracancerous tissues (Fig. 1B; P<0.001). These findings collectively indicated the increased expression of ISG20L2 in LUAD, suggesting its involvement in LUAD pathogenesis.
Furthermore, we conducted ROC curve analysis to assess the diagnostic value of ISG20L2 in LUAD. The results indicated that ISG20L2 exhibited a certain level of accuracy (AUC=0.829) in predicting LUAD (Fig. 1C). Additionally, we examined the associations between ISG20L2 expression and various clinical characteristics in LUAD patients. However, no significant correlations were observed between ISG20L2 expression and factors such as gender, age, tumor size, T stage, N stage, M stage, pathological grade, and TNM stage (Table 1).
To evaluate the prognostic significance of ISG20L2 expression at the protein level, we analyzed the relationship between ISG20L2 expression and patient prognosis using IHC staining results. Our analysis revealed that low ISG20L2 expression was associated with a better prognosis (Fig. 1D; P<0.05). Moreover, we employed the Cox regression model to assess the link between ISG20L2 expression and patient survival (Table 2). Univariate analysis demonstrated that ISG20L2 expression, age, N stage, and TNM stage were factors influencing the overall survival of lung adenocarcinoma patients. However, multivariate analysis indicated that these factors were not independent risk factors for the overall survival of lung adenocarcinoma patients.
Overall, our findings suggest that ISG20L2 is upregulated in LUAD and may play a role in its pathogenesis. Furthermore, ISG20L2 expression shows diagnostic potential and is associated with patient prognosis in LUAD.
Table 1.The relationship between the protein expression of ISG20L2 and clinicopathological characteristics in patients with lung adenocarcinoma
|
variables
|
ISG20L2 expression
|
total
|
p value
|
r value
|
|
low
|
high
|
sex
|
|
|
|
|
0.143
|
0.162
|
|
Male
|
21
|
18
|
39
|
|
|
|
Female
|
21
|
35
|
56
|
|
|
age
|
|
|
|
|
1
|
-0.009
|
|
≤60
|
21
|
27
|
48
|
|
|
|
>60
|
21
|
26
|
47
|
|
|
Tumor_size
|
|
|
|
|
0.512
|
0.071
|
|
≤4cm
|
29
|
32
|
61
|
|
|
|
>4cm
|
12
|
18
|
30
|
|
|
T
|
|
|
|
|
0.124
|
0.184
|
|
Ⅰ
|
12
|
8
|
20
|
|
|
|
Ⅱ-Ⅳ
|
27
|
44
|
71
|
|
|
N
|
|
|
|
|
0.061
|
0.21
|
|
N0
|
25
|
20
|
45
|
|
|
|
N1/N2/N3
|
17
|
32
|
49
|
|
|
M
|
|
|
|
|
1
|
0.092
|
|
M0
|
42
|
52
|
94
|
|
|
|
M1
|
0
|
1
|
1
|
|
|
grade
|
|
|
|
|
0.569
|
0.071
|
|
Ⅰ-Ⅱ
|
37
|
44
|
81
|
|
|
|
Ⅲ
|
5
|
9
|
14
|
|
|
TNM
|
|
|
|
|
0.086
|
0.193
|
|
Ⅰ
|
20
|
15
|
35
|
|
|
|
Ⅱ-Ⅳ
|
22
|
37
|
59
|
|
|
r:Correlation Coefficient, P < 0.05 was considered statistically significant.
Table 2. Univariate and multivariate analyses of the factors correlated with Overall survival of lung cancer patients
variables
|
Univariate analysis
|
Multivariate analysis
|
|
HR
|
95%CI
|
p value
|
HR
|
95%CI
|
p value
|
|
|
Lower limit
|
Upper limit
|
|
|
|
Upper limit
|
|
expression
|
1.706
|
1.005
|
2.895
|
0.0477
|
1.35
|
0.78
|
2.32
|
0.282
|
sex
|
0.991
|
0.593
|
1.656
|
0.971
|
|
|
|
|
age
|
1.953
|
1.161
|
3.284
|
0.0117
|
1.47
|
0.86
|
2.53
|
0.16
|
Tumor_size
|
1.166
|
0.673
|
2.019
|
0.585
|
|
|
|
|
T
|
1.838
|
0.927
|
3.645
|
0.0814
|
|
|
|
|
N
|
3.369
|
1.929
|
5.884
|
0.0000197
|
2.68
|
0.95
|
7.55
|
0.0617
|
M
|
1.343
|
0.185
|
9.734
|
0.77
|
|
|
|
|
grade
|
1.331
|
0.654
|
2.706
|
0.43
|
|
|
|
|
TNM
|
2.979
|
1.639
|
5.412
|
0.000341
|
1.11
|
0.37
|
3.38
|
0.853
|
HR: Hazard Ratio, CI: Confidence Interval. P < 0.05 was considered statistically significant.
3.2 GO and KEGG Enrichment Analyses
To gain a better understanding of the biological processes and pathways involved in lung cancer, we performed GO functional enrichment analysis and KEGG pathway analysis. The GO functional enrichment analysis revealed that the primary biological processes (BP) involved extracellular matrix organization, extracellular structure organization, and external encapsulating. The cellular component (CC) was primarily enriched in collagen-containing extracellular matrix, membrane raft, and membrane microdomain. The molecular function (MF) was mainly involved in glycosaminoglycan binding, extracellular matrix structural constituent, and heparin binding (Fig. 2A).
The KEGG pathway enrichment analysis identified several pathways related to lung cancer, including cytokine-cytokine receptor interaction, protein digestion and absorption, IL-17 signaling pathway, and lipid and atherosclerosis (Fig. 2B). These findings suggest that alterations in extracellular matrix organization and cytokine signaling may play a critical role in the development and progression of lung cancer.
These results provide valuable insights into the biological processes and pathways associated with lung cancer, which may aid in the development of more effective diagnostic and therapeutic strategies for this disease.
3.3 ISG20L2 overexpression promotes proliferation, migration, and invasion of A549 cells but without affecting apoptosis
To assess the efficiency of ISG20L2 overexpression, Western blot analysis was performed in both the pcDNA3.1 and pcDNA3.1-ISG20L2 groups. The results showed a significant increase in ISG20L2 expression in the pcDNA3.1-ISG20L2 group compared to the pcDNA3.1 group (Fig. 3A; P<0.001).
To further investigate the impact of ISG20L2 on the biological function of A549 cells, a CCK-8 assay was conducted to measure cell proliferation. The results demonstrated a significant increase in cell proliferation in the pcDNA3.1-ISG20L2 group after 48 hours compared to the pcDNA3.1 group (Fig. 3B; P<0.01). However, no significant difference was observed in apoptosis between the two groups following ISG20L2 overexpression (Fig. 3C,P> 0.05). These findings suggest that ISG20L2 overexpression enhances the growth rate of A549 cells in vitro but does not affect cell apoptosis.
To evaluate the migratory and invasive abilities of A549 cells, transwell assays were performed. The results indicated that cell migration and invasion were significantly increased in the pcDNA3.1-ISG20L2 group compared to the pcDNA3.1 group (Fig. 4A; Fig. 4B; P<0.05). These findings suggest that ISG20L2 overexpression promotes the migration and invasion of A549 cells.
Collectively, our results demonstrate that ISG20L2 overexpression enhances the proliferative capacity, migration, and invasion of A549 cells, indicating its potential role in promoting the progression of lung adenocarcinoma.
3.4 ISG20L2 knockdown decreases proliferation, migration, and invasion of A549 cells but without affecting apoptosis
To further investigate the effects of ISG20L2 on LUAD cells, ISG20L2 knockdown was performed, and the transfection efficiency was measured using Western blot analysis. The results revealed a reduction in ISG20L2 expression in the si-ISG20L2 group compared to the NC group (Fig. 3A; P<0.001).
Subsequently, a CCK-8 assay was conducted to evaluate the cell proliferative capacity. The results indicated a significant decrease in cell proliferation in the si-ISG20L2 group compared to the NC group (Fig. 5A; P<0.01). Flow cytometry analysis demonstrated no difference in apoptosis rate between the NC group and the si-ISG20L2 group (Fig. 5B,P > 0.05). These findings suggest that ISG20L2 knockdown reduces the proliferative ability of A549 cells, while similar to the results observed with ISG20L2 overexpression, ISG20L2 knockout does not affect apoptosis.
Furthermore, transwell assays were performed to assess cell migration and invasion. The si-ISG20L2 group exhibited significantly decreased migration and invasion abilities compared to the NC group (Fig. 5C; Fig. 5D; P<0.05). In summary, the aforementioned experiments demonstrate that ISG20L2 knockdown decreases the migration and invasion of A549 cells.
Taken together, these findings suggest that ISG20L2 plays a crucial role in promoting the proliferative capacity, migration, and invasion of LUAD cells, highlighting its potential as a target for therapeutic interventions in lung adenocarcinoma.