Clin Exp Otorhinolaryngol.  2022 Nov;15(4):372-379. 10.21053/ceo.2022.00150.

The Expression of Defensin-Associated Genes May Be Correlated With Lymph Node Metastasis of Early-Stage Tongue Cancer

• Lee DY
• Hah JH
• Jeong WJ
• Chung EJ
• Kwon TK
• Ahn SH
• Sung MW
• Kwon SK

• KMID: 2536536
• DOI: http://doi.org/10.21053/ceo.2022.00150

Abstract

Objectives
. We aimed to assess the genetic differences between cases of early-stage tongue cancer that were positive or negative for lymph node metastasis.
Methods
. In total, 35 cases of tongue cancer with RNA sequencing data were enrolled in this study. The gene expression profile of the following two groups was compared: N0 group (T stage 1 or 2 with N0 stage) and N+ group (T stage 1 or 2 with N+ stage). Using the R and limma packages in the Bioconductor program, we extracted the differentially expressed genes (DEGs). Gene ontology and pathway enrichment analysis were performed using the Database for Annotation, Visualization and Integration Discovery (DAVID) online tool. Immune cell infiltration was analyzed using the CIBERSORT online program. Immunochemical staining of the cancer tissue was evaluated and The Cancer Genome Atlas (TCGA) data were analyzed to validate the identified DEGs.
Results
. No significant differences were found in the infiltration of 22 types of immune cells. Among a total of 51 identified DEGs, 14 genes were significantly upregulated, while 37 genes were significantly downregulated (P<0.01; fold change >2). Pathway analysis revealed significant associations with the arachidonic acid metabolism-related pathway, calcium signaling, and the muscle contraction pathway. The following DEGs were the most significantly different between the two groups: DEFB4A, SPRR2B, DEFB103B, SPRR2G, DEFB4B, and FAM25A. TCGA data showed that DEFB4A and DEFB103B were more highly expressed in the N0 group than in the N+ group, although the difference did not achieve statistical significance. Immunochemical staining of cancer tissue revealed significantly higher expression of defensin in the N0 group.
Conclusions
. Defensin (DEFB4A, DEFB103B, DEFB4B) may be a novel biomarker for early regional metastasis in T1/2 tongue cancer.

Figure

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  Fig. 1. Differentially expressed genes (DEGs). Among 51 DEGs, DEFB4A, SPRR2B, DEFB103B, SPRR2G, DEFB4B, and FAM25A were the most significantly downregulated genes.

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  Fig. 2. Heatmap.

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  Fig. 3. Analysis of immune cell infiltration. Immune cell infiltration was analyzed using the CIBERSORT program. No significant differences were observed in the 22 different types of immune cells including macrophage and dendritic cells (shadow) (A) and the ratio of M1 to M2 macrophages (B). NK, natural killer.

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  Fig. 4. The Cancer Genome Atlas data analysis. Although statistical significance was not reached, the N+ group showed lower levels of DEFB103B and DEFB4A. NS, not significant.

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  Fig. 5. Immunohistochemical (IHC) staining of beta-defensin. (A) The intensity of defensin staining was measured and compared using ImageJ software. The independent t-test revealed that the two groups showed significant differences (P<0.01). (B) Representative images of defensin staining of each group (upper, ×100 magnification; lower, ×200 magnification). The composition of defensin in the cancerous epithelium was significantly higher in the N0 group compared to the N+ group.

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