Tumor necrosis factor α promotes HEp-2 cell proliferation via toll-like receptor 4 and NF-κB signaling pathways

Abstract

Laryngeal carcinoma is a serious, life-threatening disease. Tumor necrosis factor α (TNF-α), a proinflammatory cytokine, has complex effects on the proliferation and growth of cancer cells. Previously, we treated a laryngeal cancer cell line (HEp-2) with TNF-α and demonstrated that this treatment suppressed polycystin-2, a transient receptor potential cation channel expression and ATP-induced Ca²⁺ release but increased HEp-2 cell proliferation and growth. However, the mechanisms and signaling pathways underlying the TNF-α effects on the HEp-2 cells were unclear. Therefore, we here used RNA-seq techniques to examine the effect of TNF-α on the gene transcript expression profile in these cells. We found that TNF-α treatment (100 ng/mL, 24 h) upregulated 2,811 genes and downregulated 1,128 genes. The IRAK1 gene encoding an effector protein downstream of toll-like receptor 4 (TLR4) was ranked 19th in the upregulated differentially expressed genes. In a gene ontology (GO) analysis, 168 GO terms were identified in the biological process domain for the upregulated differentially expressed genes, and cell cycle and DNA replication functions were enriched. In a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, TNF-α treatment enhanced the NF-κB pathway in HEp-2 cells. Moreover, both the transcript and protein expression levels of TLR4 as well as the expression of genes encoding downstream TLR4 effectors were significantly increased in TNF-α-treated HEp-2 cells. We concluded that TNF-α increased HEp-2 cell proliferation and growth likely via enhancing TLR4- and NF-κB-associated signaling pathways and that TNF-α may play an important role in the development of laryngeal cancer.

Keywords: NF-κ; RNA-seq; Tumor necrosis factor α; human laryngeal squamous cell carcinoma; proliferation; toll-like receptor 4.

Figure 1

Differentially expressed genes and hierarchical clustering analysis in HEp-2 cells. A. The top 20 upregulated differentially expressed genes. B. Heat map showing the expression of the differentially expressed genes and hierarchical clustering analysis following TNF-α (100 ng/mL) and phosphate-buffered saline (PBS) treatment in HEp-2 cells.

Figure 2

Gene ontology (GO) enrichment analysis and protein-to-protein interaction networks in HEp-2 cells. A. The top 20 identified GO terms in the biological process category for the upregulated differentially expressed genes. B, C. Protein-to-protein interaction networks of cell cycle- and DNA replication-related genes in the upregulated differentially expressed genes.

Figure 3

TNF-α-induced changes in the NF-κB signaling pathway in HEp-2 cells. HEp-2 cells were treated with TNF-α (100 ng/mL) for 24 h. Upregulated genes are shown in red.

Figure 4

Transcript expression profiles of toll-like receptor 4 (TLR4)- and NF-κB-associated signaling pathways and the effect of TNF-α on TLR4 protein expression in HEp-2 cells. A. Heat map showing transcript expression of TLR4- and NF-κB-associated signaling pathways. B. Representative immunoblot images (upper) and summary data (lower) showing TLR4 protein expression levels in HEp-2 cells pretreated with TNF-α (100 ng/mL) o r phosphate-buffered saline (PBS, control) for 24 h. GAPDH was used as a loading control. Values are shown as the mean ± SEM (n = 3); *P < 0.05, control vs. TNF-α treatment.