Long non-coding RNA expression profiles and related regulatory networks in areca nut chewing-induced tongue squamous cell carcinoma

Abstract

Areca nut chewing is an important risk factor for developing tongue squamous cell carcinoma (TSCC), although the underlying molecular mechanism is unknown. To determine the potential molecular mechanisms of areca nut chewing-induced TSCC, the present study performed whole-genome detection with five pairs of TSCC and adjacent normal tissues, via mRNA- and long non-coding (lnc)RNA-gene chip analysis. A total of 3,860 differentially expressed genes were identified, including 2,193 lncRNAs and 1,667 mRNAs. Gene set-enrichment analysis revealed that the differentially expressed mRNAs were enriched in chromosome 22q13, 8p21 and 3p21 regions, and were regulated by nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRFs). The results of ingenuity pathway analysis revealed that these mRNAs were significantly enriched for inflammatory immune-related signaling pathways. A co-expression network of mRNAs and lncRNAs was constructed by performing weighted gene co-expression network analysis. The present study focused on NF-κB-, IRF- and Th cell-signaling pathway-related lncRNAs and the corresponding mRNA-lncRNA regulatory networks. To the best of our knowledge, the present study was the first to investigate differential mRNA- and lncRNA-expression profiles in TSCCs induced by areca nut chewing. Inflammation-related mRNA-lncRNA regulatory networks driven by IRFs and NF-κB were identified, as well as the Th cell-related signaling pathways that play important carcinogenic roles in areca nut chewing-induced TSCC. These differentially expressed mRNAs and lncRNAs, and their regulatory networks provide insight for further analysis on the molecular mechanism of areca nut chewing-induced TSCC, candidate molecular markers and targets for further clinical intervention.

Keywords: areca nut; expression profile; long non-coding RNA; network; tongue squamous cell carcinoma.

Figure 1.

Differentially expressed molecular heat maps of TSCC and adjacent normal tissues. (A) Heat map of differentially expressed mRNAs and lncRNAs. (B) Heat map of differentially expressed mRNAs. (C) Heat map of differentially expressed lncRNAs. Fold-change ≥2 and P<0.05. T represents TSCC tissue and N represents adjacent normal tissue. Red indicates upregulated genes and green indicates downregulated genes. TSCC, tongue squamous cell carcinoma; lncRNA, long non-coding RNA.

Figure 2.

GSEA revealed that differentially expressed genes in TSCC and adjacent normal tissues are enriched in chromosome 22q13. (A) GSEA gene sets demonstrated that several genes located in the 22q13 segment were upregulated in TSCC. (B) Heat map of differentially expressed long non-coding RNAs (asterisks) and mRNAs from the 22q13 segment. GSEA, gene set-enrichment analysis; TSCC, tongue squamous cell carcinoma.

Figure 3.

WGCNA-constructed mRNA-lncRNA co-expression network in TSCC. (A) Topological overlap matrix heat map of differentially expressed lncRNAs and mRNAs, which demonstrated that the network exhibits a scale-free topology. Red represents a lower overlap, while yellow represents a higher overlap. The top and left sides of the heat map are hierarchical cluster trees, with different branches of the cluster tree representing different gene modules, while the corresponding colors represent different modules. (B) Co-expression network of differentially expressed mRNAs and lncRNAs in TSCC and adjacent normal tissues, obtained via WGCNA and visualized using Cytoscape software. The network consisted of 913 nodes, including 538 lncRNAs and 375 mRNAs, and 60,223 linkages (mRNA-lncRNA associations). The topological-overlap value was higher than the threshold of 0.40. WGCNA, weighted gene co-expression network analysis; lncRNA, long non-coding RNA; TSCC, tongue squamous cell carcinoma.

Figure 4.

Prediction of IRF- and NF-κB-regulated mRNAs via GSEA, screening related lncRNAs and construction of regulatory networks. (A) The upstream regulators of differentially expressed mRNAs were predicted via GSEA. IRF- and NF-κB-regulated mRNAs were the most significantly enriched. (B) mRNA regulatory networks driven by IRFs and NF-κB. (C) The co-expression heat map of IRF-regulated mRNAs and lncRNAs (asterisks). In the heatmap, some mRNAs were derived from the reference gene set that was regulated by IRFs in the GSEA database, whereas others were significantly associated with IRFs in the WGCNA-constructed co-expression network. The lncRNAs that were significantly associated with IRFs in the WGCNA-constructed co-expression network are presented. (D) The co-expression heat map of NF-κB-regulated mRNAs and lncRNAs (asterisks). In the heatmap, some mRNAs were derived from the reference gene set that was regulated by NF-κB in the GSEA database, whereas others were significantly associated with NF-κB in the WGCNA-constructed co-expression network. The lncRNAs that were significantly associated with NF-κB in the WGCNA-constructed co-expression network are presented. (E) mRNA-lncRNA regulatory networks driven by IRFs and NF-κB. Yellow represents IRFs or NF-κB; green represents mRNAs derived from the reference gene set that was regulated by IRFs or NF-κB in the GSEA database; blue represents lncRNAs significantly associated with IRFs or NF-κB in the WGCNA-constructed co-expression network and red represents mRNAs significantly associated with IRFs or NF-κB in the WGCNA-constructed co-expression network. IRF, interferon regulatory factor; NF-κB, nuclear factor kappa B; GSEA, gene set-enrichment analysis; lncRNA, long non-coding RNA; WGCNA, weighted gene co-expression network analysis.

Figure 5.

Network of lncRNAs involved in regulating the Th cell activation pathway in tongue squamous cell carcinoma. The lncRNAs were derived from the co-expression network and had similar expression trends compared with the mRNAs in the Th cell activation pathway. The triangles represent lncRNAs. Round and oval represent mRNAs. Red and pink represent upregulation, green represents downregulation and white represents no difference in expression. lncRNA, long non-coding RNA.