Differentially expressed genes related to lymph node metastasis in advanced laryngeal squamous cell cancers

Abstract

Understanding the molecular mechanisms and gene expression in laryngeal squamous cell carcinoma (LSCC) may explain its aggressive biological behavior and regional metastasis pathways. In the present study, patients with locally advanced LSCC tumors were examined for differential gene expression in the normal mucosa (non-tumoral mucosa), tumors and lymph node tissues. The aim was to identify possible predictive genes for lymph node metastasis. A total of 16 patients who had undergone total laryngectomy with neck dissection for advanced LSCC were randomly selected from a hospital database: Eight of the patients had lymph node metastasis (Group 1) and the other eight patients did not have metastasis (Group 2). Overall survival (OS), disease-free survival (DFS) and disease-specific survival (DSS) were analyzed. For each patient, paraffin-embedded tissue samples were collected from non-tumoral mucosa, tumoral lesions and lymph node tissues. RNA was extracted from the tissue samples and used for complementary DNA synthesis, and microarray analysis was subsequently performed on each sample. Gene expression levels were determined in each specimen, and Groups 1 and 2 were compared and statistically analyzed. The microarray results for lymph node metastasis-positive and -negative groups, indicated the differential expression of 312 genes in the lymph nodes, 691 genes in the normal mucosal tissue and 93 genes in the tumor tissue. Transgelin (TAGLN) and cofilin 1 (CFL1) were identified as possible target genes and validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR results for TAGLN and CFL1 supported the microarray data. OS, DFS and DSS times were longer in Group 2 than in Group 1 (P=0.002, 0.015 and 0.009, respectively). In addition, TAGLN and CFL1 were associated with DFS and DSS. On the basis of these results, it is suggested that TAGLN and CFL1 expression may play an important role in the pathogenesis of regional metastasis and poor prognosis in advanced LSCC.

Keywords: cofilin-1; gene expression profile; laryngeal squamous cell cancer; microarray; tansgelin.

Figure 1.

Kaplan-Meier survival curves of the patients. (A) Overall survival, (B) disease-free survival and (C) disease-specific curves. Lower (green) curves represent patients with LNM (Group 1) and upper (blue) curves represent patients without LNM (Group 2). LNM, lymph node metastasis.

Figure 2.

Differential expression analysis between metastasis-negative and -positive patients in tumor, lymph and normal mucosal tissues. (A) Volcano plot and (B) hierarchical gene analysis. In the volcano plot, the red circles indicate cofilin 1 and transgelin genes. Fold change values are for the metastasis-negative group relative to the metastasis-positive group. Transgelin is indicated with purple circle and cofilin 1 indicated with black circle.

Figure 3.

Differential expression analysis between metastasis-negative and -positive patients in lymph tissues. (A) Volcano plot and (B) hierarchical gene analysis. In the volcano plot, the black circles indicate cofilin 1 and purple circle indicate transgelin genes. Fold change values are for the metastasis-negative group relative to the metastasis-positive group.

Figure 4.

Differential expression analysis between metastasis-negative and -positive patients in normal mucosal tissues. (A) Volcano plot and (B) hierarchical gene analysis. In the volcano plot, the black circles indicate cofilin 1 and purple circle indicate transgelin genes. Fold change values are for the metastasis–negative group relative to the metastasis-positive group (‘Variable charge, X-linked 2’ has two identical point for two different probes. Please see Table SIII).

Figure 5.

Differential expression analysis between metastasis-negative and -positive patients in tumor tissues. (A) Volcano plot and (B) hierarchical gene analysis. Fold change values are for the metastasis-negative group relative to the metastasis-positive group.

Figure 6.

Differentially expressed genes in the tumor, lymph and normal mucosal tissues of patients with metastasis-negative and -positive laryngeal squamous cell carcinoma. (A) Cofilin 1 and (B) transgelin RNA expression levels.