Differential Expression of ATⅡ-associated genes in Patients With NSCLC
We first explored the expression levels of 8 ATⅡ-associated genes in lung cancer tissues and normal para-carcinoma tissues using the Oncomine database, the mRNA expression levels of AQP4, CLDN18, FOXA2, NKX2-1, PGC and SFTPB, SFTPC, SFTPD were all remarkably reduced in lung cancer tissues in multiple datasets (Figure 1). Furthermore, we used the GEPIA dataset to compare the mRNA expressions of the ATⅡ-associated genes in both 483 LUAD and 347 normal tissues and 486 LUSC, and 338 normal tissues. Our results indicated that AQP4, CLDN18, PGC and SFTPB, SFTPC, SFTPD were low expression in LUAD tissues, and the AQP4, CLDN18, FOXA2, NKX2-1, PGC and SFTPB, SFTPC, SFTPD were lower expression in the LUSC tissues (Figure 2). We also contrast the relative expression levels of eight ATⅡ-associated genes in LUAD and LUSC tissues. The results reveal the highest expression of gene in LUAD and LUSC is SFTPB (Figure 3).
According to the above results, we considered that transcriptional expressions of AQP4, CLDN18, FOXA2, NKX2-1, PGC SFTPB, SFTPC, and SFTPD were low-expression in patients with NSCLC.
Correlation Between mRNA Expression of Different ATⅡ-associated genes and Tumor Stages of NSCLC Patients
Lung cancer is divided into four stages according to the disease progression. As the condition develops, the patient's physiology and physical condition will also constantly change. Therefore, we assessed the correlation between the expression of ATⅡ-associated genes and the patients' pathological cancer stages of LUAD and LUSC patients by using GEPIA. We found that the a significant correlation between the expression of all eight ATⅡ-associated genes and pathological stage of NSCLC : AQP4(P = 1.81e-06), CLDN18(P = =4.64e -06), FOXA2(P = 0.000128), NKX2-1(P =0.000756), PGC(P = 3.08e-07),SFTPB(P =3.33e-07)/SFTP C(P = 1.4e-08)and SFTP D (P = 1.54e-07),band NSCLC patients who were in more advanced cancer stages were all almost inclined to express higher mRNA expression of ATⅡ-associated genes.(Figure 4). These data suggested that the 8 ATⅡ-associated might play a significant role in the tumorigenesis and progression of NSCLC.
Prognostic Features of ATⅡ-associated genes in Patients With Lung Cancer
To analyze the prognostic values of ATⅡ-associated genes in NSCLC patients, we assessed the correlation between these genes and prognosis using Kaplan-Meier plotter (Table 1). The survival of patients including overall survival (OS), progression-free survival (FP), and post-progression survival (PPS) (p < 0.05) (Figure 5 ).The results revealed that the decreased AQP4 (HR = 0.74, p = 0.00024), CLDN18 (HR = 0.76, p = 1.9e-05), FOXA2 (HR = 0.63, p = 1.6e-12), NKX2-1 (HR = 0.67, p = 4.9e-10), PGC (HR = 0.69, p = 1e-08), SFTPB (HR = 0.67, p = 6.3e-10), SFTPC (HR = 0.81, p = 0.0014) and SFTPD (HR = 0.66, p = 1.6e-10) mRNA levels were expressively associated with low OS. Besides, the low expression of CLDN18 (HR = 0.72, p = 0.00091), FOXA2 (HR = 0.68, p = 6.7e-05), NKX2-1 (HR = 0.81, p = 0.031), PGC (HR = 0.7, p = 0.00024), SFTPB (HR = 0.82, p = 0.048), SFTPC (HR = 0.82, p = 0.04) and SFTPD (HR = 0.68, p = 6.2e-05) were significantly related to a reduced FP. Finally, low CLDN18 (HR = 0.98, p = 0.032), FOXA2 (HR = 0.74, p = 0.021) and SFTPD (HR = 0.96, p = 0.021) mRNA expression apparently led to a short PPS. However, no significant difference was found between the ATⅡ-associated genes and Disease-free survival (DFS) in NSCLC patients. (Table 1).
Table 1
The Relationship Between the Expression Level of ATII-associated genes and Prognosis in Patients with NSCLC
ATII-assciated genes
|
Kaplan-Meier Plotter (Logrank p)
|
GEPIA (Logrank p)
|
OS
|
FP
|
PPS
|
DFS
|
AQP4
|
0.00024
|
0.056
|
0.4
|
0.74
|
CLDN18
|
1.90E-05
|
0.00091
|
0.032
|
0.69
|
FOXA2
|
1.60E-12
|
6.70E-05
|
0.021
|
0.81
|
NKX2-1
|
4.90E-10
|
0.031
|
0.12
|
0.13
|
PGC
|
1.00E-08
|
0.00024
|
0.59
|
0.22
|
SFTP-B
|
6.30E-10
|
0.048
|
0.38
|
0.42
|
SFTP-C
|
0.0014
|
0.04
|
0.11
|
0.29
|
SFTP-D
|
1.60E-10
|
6.20E-05
|
0.021
|
0.4
|
Genetic Alteration, Expression and Protein/Gene Interaction Analyses of ATⅡ-associated genes in Patients With NSCLC
Epigenetic alteration plays a vital role in early malignancies, so a comprehensive analysis of the molecular characteristics of ATⅡ-associated genes was further performed on the LUAD and LUSC samples, respectively. We used the cBioPortal online tool to analyze the ATⅡ-associated genes alterations for LUAD (TCGA, Pan- Cancer Atlas) and LUSC (TCGA, Pan-Cancer Atlas). As a group, two or more alterations were detected in different subtypes of NSCLC, and the 8 ATⅡ-associated genes were varied in 273 samples out of 1053 patients with NSCLC (26%) (Figure 6A). Moreover, the mutation rates of AQP4, CLDN18, FOXA2, NKX2-1, PGC and SFTPB, SFTPC, SFTPD were 3, 5, 2.4, 9, 2.8, 1.8, 5, and 1.1% of the investigated lung cancer samples, respectively (Figure 6A).
Moreover, a PPI network analysis of ATⅡ-related genes was conducted with STRING. The results in Figure 6B exposed that the (deleted inmalignant brain tumors) DMBT1 gene which is a candidate tumor suppressor gene recently discovered in recent years was closely connected with ATⅡ-associated genes alterations (Figure 6B). Besides, some genes that play an important role in immune response regulation, blood cell proliferation, defense mechanisms, and acute phase response genes are also significantly connected with ATⅡ-associated genes alterations. including Microfibril-associated glycoprotein 4 (MFAP4), Pulmonary surfactant-associated protein A1 (SFTPA1) (Figure 6B). The GeneMANIA results also revealed that the functions of the differentially expressed ATⅡ-associated genes and their associated molecules (such as, Leucine-rich repeat kinase 2 (LRRK2), lysosomal-associated membrane protein 3 (LAMP3), Cathepsin E (CTSE), ATP-binding cassette transporter A3 (ABCA3), forkhead box F1(FOXF1), and Napsin A (NAPSA)) were primarily related to lung development, late endosome, aspartic-type peptidase activity (Figure 6C).
Immune Cell Infiltration of ATⅡ-associated genes in Patients With NSCLC
Immune cell level is associated with the proliferation and progression of the cancer cell. In this study, to verify ATⅡ-associated genes have been involved in cancer-related inflammation and the infiltration of immune cells, thus affecting the clinical outcome of NSCLC patients, we use the TIMER database to provide a comprehensive analysis of the correlation between eight ATⅡ-associated genes and immune cell infiltration (Figure 7, Figure 8). As the result shows, the expression levels of SFTPC and CLDN18 and the infiltration of neutrophils, B cells, macrophages, CD4+ T cells, dendritic cells, and CD8+ T cells have a positive correlation both in LUSC and LUAD (P < 0.05). In LUSC, all ATⅡ-associated genes (including AQP4, FOXA2, NKX2-1, PGC, SFTPB, SFTPD, CLDN18, SFTPC) were positively associated with the infiltration of six immune cell types (neutrophils, B cells, macrophages, CD4+ T cells, dendritic cells, and CD8+ T cells; all P < 0.05) and these genes also were positively related to the infiltration of B cells in LUAD (P < 0.05). Besides, the expression levels of AQP4, FOXA2, NKX2-1, SFTPB, CLDN18, SFTPC had a positive relation to the infiltration of CD8+T cells (P < 0.05) and the expression of NKX2-1, CLDN18, SFTPD, SFTPC had a positive relation to the infiltration of CD4+T cells (P < 0.05).