Differential Expression of the TLR4 Gene in Pan-Cancer and Its Related Mechanism

Abstract

Previous studies have revealed the relationship between toll-like receptor 4 (TLR4) polymorphisms and cancer susceptibility. However, the relationship between TLR4 and prognosis and immune cell infiltration in pan-cancer patients is still unclear. Through the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases, the distinct expression of the TLR4 gene in 24 tumors and normal tissues was analyzed. Univariate Cox proportional hazards regression analysis was used to identify the cancer types whose TLR4 gene expression was related to prognosis. The relationship between TLR4 and tumor cell immune invasion was studied. Spearman's rank correlation coefficient was used to analyze the relationship among TLR4 and immune neoantigens, tumor mutation burden (TMB), microsatellite instability (MSI), DNA repair genes, and DNA methylation. Gene Set Enrichment Analysis (GSEA) was used to identify the tumor-related pathways that the TLR4 gene was highly expressed in; the expression of the TLR4 gene was verified with the Human Protein Atlas (HPA) database. Low expression of TLR4 was associated with an inferior prognosis in kidney renal clear cell carcinoma (KIRC), skin cutaneous melanoma (SKCM), and uterine corpus endometrial carcinoma (UCEC), while high expression was related to a poor prognosis in head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), stomach adenocarcinoma (STAD), and testicular germ cell tumor (TGCT). The expression of TLR4 was negatively correlated with the expression of B cells in STAD. The expression of TLR4 was positively correlated with the infiltration of B cells, CD4 and CD8 T cells, neutrophils, macrophages, and dendritic cells in STAD, KIRC, UCEC, TGCT, and SKCM. The expression of the TLR4 gene in KIRC, SKCM, STAD, TGCT, and UCEC was highly correlated with inducible T-cell costimulator (ICOS), cytotoxic T lymphocyte-associated molecule 4 (CTLA4), and CD28 immune checkpoints. Spearman's rank correlation coefficient showed that the expression of TLR4 gene was significantly correlated with TMB in STAD and UCEC and was prominently correlated with MSI in TGCT, STAD, and SKCM. The expression of the TLR4 gene was highly correlated with MLH1, MSH2, and MSH6 in KIRC, SKCM, and STAD. The expression of the TLR4 gene was remarkably correlated with the methyltransferases DNA methyltransferase 2 (DNMT2) and DNA methyltransferase 3-beta (DNMT3B) in SKCM and STAD. Enrichment analysis showed that TLR4 was highly expressed in the chemokine signaling pathway and the cell adhesion molecule and cytokine receptor interaction pathway. In summary, the expression of TLR4 is linked to the prognosis of KIRC, SKCM, STAD, TGCT, and UCEC patients and the level of immune infiltration of CD4, CD8 T cells, macrophages, neutrophils, and dendritic cells.

Keywords: bioinformatics; immune cell infiltration; pan-cancer; prognosis; toll-like receptor 4.

FIGURE 1

Comprehensive analysis of the expression of Toll-like receptor 4 (TLR4) gene in various databases. The Genotype-Tissue Expression (GTEx) data set showed that the TLR4 gene was basically not expressed in bone marrow or 31 other tissues and was most highly expressed in the spleen (A). The CCLE database (n = 1,019) showed that the TLR4 gene expression was lowest in salivary glands and highest in the central nervous system (B). There are few normal tissue data in The Cancer Genome Atlas (TCGA) database (C). The GTEx database (n = 6,678) and the tumor data in TCGA database (n = 9,498) are compared and displayed (D). Compared with normal tissues, TLR4 gene expression is low in ACC, BLCA, BRCA, CESC, CHOL, HNSC, KIRP, LUAD, READ, HCA, UCEC, and UCS. In some data sets, COAD, ESCA, GBM, KICH, LGG, LIHC, PAAD, SKCM, STAD, and TGCT have high expressions. PFI showed that TLR4 was notably related to the prognosis of CHOL (p = 0.015), KIRC (p = 2.6e-5), and PRAD (p = 0.025) (E). OS displayed that TLR4 was markedly correlated with the prognosis of KIRC (p = 0.0018), SKCM (p = 0.0064), STAD (p = 0.038), TGCT (p = 0.03), and UCEC (p = 0.049) (F). DSS reflected that TLR4 was observably correlated with the prognosis of KIRC (p = 0.00019), SKCM (p = 0.0094), and UCEC (p = 0.02) (G). The prognosis of DFI showed that TLR4 was dramatically correlated with the prognosis of HNSC (p = 0.0089) and PRAD (p = 0.013) (H). A p-value <0.05 was considered statistically significant. The symbols “^∗,” “^∗∗,” and “^∗∗∗” refer to p-values <0.05, <0.01, and <0.001, respectively.

FIGURE 2

Survival curve of cancer types with significant correlation between TLR4 gene expression and prognosis. The low expression of TLR4 was related to the low survival rate of KIRC (p < 0.0001) (C,G,I), SKCM (p = 0.00023) (D,J), UCEC (p = 0.00079) (E,M), and CHOL (p = 0.0048) (F). High TLR4 expression is related to low survival rates of HNSC (p = 0.001) (A), PRAD (p = 0.0024) (B,H), STAD (p = 0.0095) (K), and TGCT (p = 0.00076) (L).

FIGURE 3

Correlation analysis of TLR4 gene expression in immune infiltration, immune checkpoint, and in 33 tumors. (A–E) The expression of TLR4 was negatively correlated with the expression of B cells in STAD, KIRC, UCEC, TGCT, and SKCM. While the expression of TLR4 was positively correlated with the infiltration of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells in STAD, KIRC, UCEC, TGCT, and SKCM. (F) The results showed that the five prognostic-related cancers of KIRC, SKCM, STAD, TGCT, and UCEC are highly positively correlated with the six immune checkpoints of ICOS, CTLA4, CD28, CD80, PDCD1LG2, and CD86 (r > 0, ***p < 0.001).

FIGURE 4

The relationship between the TLR4 gene expression and immune score, neoantigens. (A) The expression of TLR4 gene in the ESTIMATE immune score was concerned with CESC (p = 0), ESCA (p = 0), HNSC (p = 0) are prominently correlated. (B) KIRC, SKCM, STAD, TGCT, and UCEC were not notably related to neoantigens. A p-value <0.05 was considered statistically significant. The symbols “*,” “**,” and “***” refer to p-values <0.05, <0.01, and <0.001, respectively.

FIGURE 5

The relationship between TLR4 gene expression and TMB, MSI, methyltransferase, and mutation frequency. (A) The expression of TLR4 gene was noticeably relevant to TMB in STAD (p = 0.046) and UCEC (p = 0.0044). (B) The expression of TLR4 gene in TGCT (p = 0.0071), STAD (p = 1.6e-05), and SKCM (p = 4.6e-05) was significantly correlated with MSI. (C) The analysis outcomes revealed that the expression of TLR4 gene was conspicuously linked to methyltransferase DNMT2 and DNMT3B in SKCM and STAD (p < 0.05) and was markedly correlated with methyltransferase DNMT2 in UCEC and KIRC (p < 0.05). TCGA analysis displayed that the mutation rates of TLR4 gene in KIRC (D), SKCM (E), STAD (F), and UCEC (G) were 0.6, 11.35, 5.95, and 7.74%, respectively.

FIGURE 6

Analysis of the TLR4 gene enrichment and DNA repair genes. (A) KEGG analysis displayed that TLR4 was highly expressed in chemokine signaling pathway (p = 0), cell adhesion molecule (p = 0), and interaction of cytokine receptor (p = 0). HALLMARK analysis showed that TLR4 is lowly expressed in MYC_TARGETS_V2 (p = 0.048) and highly expressed in KRAS signaling pathway (p = 0) and inflammatory response (p = 0) pathway. (B) The TLR4 gene expression was highly linked to DNA repair genes MLH1, MSH2, MSH6, and PMS2 in KIRC and STAD and MLH1, MSH2, and MSH6 in SKCM (**p < 0.001); mildly correlated with MLH1, MSH2, and PMS2 in UCEC (*p < 0.001) 05); and showed a moderate correlation with EPCAM (**p < 0.01). A p-value <0.05 was considered statistically significant.

FIGURE 7

HPA database verifies the expression of TLR4 gene in seven tumors. The expression of TLR4 gene in LIHC (A), COAD (B), OV (C), PRAD (D), BRCA (E), PAAD (F), and UCEC (G) is significantly higher than that in the corresponding normal tissues.

FIGURE 8

Immunohistochemistry verifies the expression of TLR4 gene in seven tumors. The expression of TLR4 gene in LIHC (A), COAD (B), OV (C), PRAD (D), BRCA (E), PAAD (F), and UCEC (G) is significantly higher than that in the corresponding normal tissues.