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. 2022 Jul 25:12:911316.
doi: 10.3389/fonc.2022.911316. eCollection 2022.

Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

Jianglin Wang  1 , Qingping Yang  2 , Mengjie Tang  3 , Wei Liu  1
Affiliations

Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

Jianglin Wang et al. Front Oncol. .

Abstract

Early diagnosis and prognosis prediction of non-small cell lung cancer (NSCLC) have been challenging. Signaling cascades involving the Wingless-type (WNT) gene family play important biological roles and show prognostic value in various cancers, including NSCLC. On this basis, this study aimed to investigate the significance of WNTs in the prognosis and tumor immunity in NSCLC by comprehensive analysis. Expression and methylation levels of WNTs were obtained from the ONCOMINE, TIMER, and UALCAN. The dataset obtained from The Cancer Genome Atlas (TCGA) was utilized for prognostic analysis. cBioPortal was used to perform genetic alterations and correlation analysis of WNTs. R software was employed for functional enrichment and pathway analysis, partial statistics, and graph drawing. TRRUST was used to find key transcription factors. GEPIA was utilized for the analysis of expression, pathological staging, etc. Correlative analysis of immune infiltrates from TIMER. TISIDB was used for further immune infiltration validation analysis. Compared with that of normal tissues, WNT2/2B/3A/4/7A/9A/9B/11 expressions decreased, while WNT3/5B/6/7B/8B/10A/10B/16 expressions increased in lung adenocarcinoma (LUAD); WNT2/3A/7A/11 expressions were lessened, while WNT2B/3/5A/5B/6/7B/10A/10B/16 expressions were enhanced in squamous cell lung cancer (LUSC). Survival analysis revealed that highly expressed WNT2B and lowly expressed WNT7A predicted better prognostic outcomes in LUAD and LUSC. In the study of immune infiltration levels, WNT2, WNT9B, and WNT10A were positively correlated with six immune cells in LUAD; WNT1, WNT2, and WNT9B were positively correlated with six immune cells in LUSC, while WNT7B was negatively correlated. Our study indicated that WNT2B and WNT7A might have prognostic value in LUAD, and both of them might be important prognostic factors in LUSC and correlated to immune cell infiltration in LUAD and LUSC to a certain extent. Considering the prognostic value of WNT2B and WNT7A in NSCLC, we validated their mRNA and protein expression levels in NSCLC by performing qRT-PCR, western blot, and immunohistochemical staining on NSCLC pathological tissues and cell lines. This study may provide some direction for the subsequent exploration of the prognostic value of the WNTs and their role as biomarkers in NSCLC.

Keywords: LUAD; LUSC; NSCLC; WNT gene family; immune infiltration; prognosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The mRNA transcription levels of the WNT gene family in LUAD and LUSC. (A) The mRNA transcription levels of WNT gene family in lung cancer (ONCOMINE). Red for overexpression, blue for downregulated expression. (B) Differential expression of WNT gene family in different tumor tissues and normal tissues (TIMER). Statistical significance of differential expression was assessed using the Wilcoxon test (p value significant codes: 0 ≤ *** < 0.001 ≤ ** < 0.01 ≤ * < 0.05 ≤. < 0.1). (C) The expression matrix plots for WNT gene family in LUAD and LUSC. Indicates the relative expression level among WNT genes.
Figure 2
Figure 2
Compared with normal tissues, the WNT gene family’s mRNA expression and methylation levels in LUAD and LUSC (UALCAN). (A) The mRNA transcription levels of the WNT gene family in LUAD and LUSC. (B) The methylation level of the WNT gene family in LUAD and LUSC. p < 0.05 was considered statistically significant.
Figure 3
Figure 3
The mRNA expression of WNT gene family in different pathological stages of LUAD and LUSC (GEPIA). (A) Correlation between WNT gene family and pathological stage in patients with LUAD. (B) Correlation between WNT gene family and pathological stage in patients with LUSC.
Figure 4
Figure 4
The overall survival (OS) curves of WNTs in LUAD and LUSC. (A) Kaplan-Meier survival analysis of WNTs in LUAD. (B) Kaplan-Meier survival analysis of WNTs in LUSC.
Figure 5
Figure 5
Univariate and multivariate Cox regression analysis of WNT gene expression and clinical characteristics and the nomogram predicting 1-y, 3-y, and 5-y survival in patients with LUAD and LUSC. (A, B, E, F) Hazard ratio and p value of constituents involved in univariate and multivariate Cox regression in LUAD (A, B) and LUSC (E, F). (C, G) Nomogram to predict the 1-y, 3-y and 5-y overall survival of LUAD (C) and LUSC (G) patients. (D, H) Calibration curve for the overall survival nomogram model in the discovery group. The dashed diagonal line represents the ideal nomogram, and the red, orange and blue represent the 1-y, 3-y and 5-y observed nomograms. (D) for LUAD. (H) for LUSC.
Figure 6
Figure 6
Genetic alterations and association analysis of the WNT gene family in NSCLC patients. (A) Genetic alterations of the WNT Gene Family in LUAD (cBioPortal). (B) Genetic alterations of the WNT Gene Family in LUSC (cBioPortal). (C, D) Correlation analysis heatmap between different WNT genes in LUAD (C) and LUSC (D). (E) Correlation analysis between different WNT genes (GEPIA).
Figure 7
Figure 7
(A–D) GO and KEGG enrichment analysis of WNT gene family. (E, F) Protein-protein interaction network of WNT gene family. (E) for STRING. (F) for Cytoscape.
Figure 8
Figure 8
Correlation of WNT genes with immune cell infiltration in LUAD and LUSC (TIMER). (A) WNT1. (B) WNT2. (C) WNT2B. (D) WNT3. (E) WNT3A. (F) WNT4. (G) WNT5A. (H) WNT5B. (I) WNT6. (J) WNT7A. (K) WNT7B. (L) WNT8A. (M) WNT8B. (N) WNT9A. (O) WNT9B. (P) WNT10A. (Q) WNT10B. (R) WNT11. (S) WNT16.
Figure 9
Figure 9
The association of WNT2B and WNT7A with immune cell infiltration (TISIDB). (A, C) The landscape of relationship between WNT2B (A) and WNT7A (C) expression and TILs in different types of cancer (red is positive correlated and blue is negative correlated). (B) Relationship between WNT2B and immune infiltration levels of B cell, CD4+ T cell, macrophage, neutrophil, dendritic cell in LUAD, and CD8+ T cell, neutrophil, dendritic cell in LUSC. (D) Relationship between WNT7A and immune infiltration levels of CD4+ T cells, macrophage, neutrophil, dendritic cell in LUAD, and B cell, CD4+ T cell, macrophage in LUSC.
Figure 10
Figure 10
Comparison of tumor infiltration levels among tumors with different SCNA for WNTs in LUAD and LUSC samples (p value significant codes: 0 ≤ *** < 0.001 ≤ ** < 0.01 ≤ * < 0.05 ≤. < 0.1). (A) WNT1. (B) WNT2. (C) WNT2B. (D) WNT3. (E) WNT3A. (F) WNT4. (G) WNT5A. (H) WNT5B. (I) WNT6. (J) WNT7A. (K) WNT7B. (L) WNT8A. (M) WNT8B. (N) WNT9A. (O) WNT9B. (P) WNT10A. (Q) WNT10B. (R) WNT11. (S) WNT16.
Figure 11
Figure 11
Validation of mRNA and protein expression levels of WNT2B and WNT7A in NSCLC. (A) Comparison of mRNA expression levels of WNTs in 20 paired NSCLC tissues and adjacent normal tissue samples by qRT-PCR. (B) Comparison of mRNA expression levels of WNTs in LUAD cell line (PC9) and LUSC cell line (NCI-H520) with human normal lung epithelial cell line (BEAS-2B). (C-F) The western blot analyses. (G–J) The immunohistochemical staining in 20 NSCLC tissues and adjacent normal tissues.
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