Downregulation of thrombospondin-1 by DNA hypermethylation is associated with tumor progression in laryngeal squamous cell carcinoma

Abstract

Thrombospondin‑1 (THBS‑1) has been demonstrated to have a complicated role in human cancer and to exert stimulatory and inhibitory effects in different types of tumors. DNA methylation, as the most frequent mechanism for gene silencing, has been widely investigated in regards to the development of tumors. However, the expression levels and methylation status of THBS‑1, and their roles in laryngeal squamous cell carcinoma (LSCC) remain to be elucidated. The present study detected downregulated THBS‑1 mRNA and protein expression levels in LSCC by using reverse transcription-quantitative polymerase chain reaction (PCR) and western blotting, while decreased expression levels of THBS‑1 mRNA and protein were significantly associated with lymph node metastasis and tumor‑node‑metastasis (TNM) stage. Furthermore, aberrant methylation of THBS‑1 was frequently observed in LSCC by methylation‑specific PCR, particularly in tumor tissues from lymph node metastasis or samples from cancer with advanced TNM stage. Furthermore, the current study demonstrated that downregulated expression of THBS‑1 in LSCC was consistent with aberrant methylation of this gene. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxy-cytidine in Hep‑2 cells induced demethylation of THBS-1, enhanced THBS‑1 expression, and inhibited the proliferative and invasive ability of Hep‑2 cells. Collectively, the results of the present study suggest that THBS‑1 may exert an inhibitory effect in the development of LSCC. Aberrant methylation was an important reason for the downregulation of THBS‑1 and was involved in the invasion and metastasis of LSCC. Demethylating agents may be effective candidates for the treatment of LSCC.

Figure 1

The mRNA and protein expression levels of THBS-1 in laryngeal squamous cell cancer tissues and corresponding adjacent normal tissues. (A) Relative mRNA levels of THBS-1 were detected by reverse transcription-quantitative polymerase chain reaction. (B) The semiquantitative analysis of the relative THBS-1 protein expression levels. (C) Representative image of western blotting analysis of THBS-1 protein. ^**P<0.01 vs. N group. N, corresponding adjacent normal tissue; T, laryngeal squamous cell cancer tissue.

Figure 2

Methylation status of THBS-1 in laryngeal squamous cell cancer tissues and corresponding adjacent normal tissues. Lane U, unmethylated bands; lane M, methylated band. N, corresponding adjacent normal tissue; T, laryngeal squamous cell cancer tissue; P, positive control; H₂O, blank control.

Figure 3

Changes in methylation status and expression of THBS-1 induced by 5-aza-dC in Hep-2 cells. (A) Methylation-specific PCR indicating the presence of unmethylated bands of THBS-1 in Hep-2 cells treated with 1 µmol/l 5-aza-dC. The presence of only methylated bands indicated complete methylation while the presence of methylated and unmethylated bands indicated a partially methylated state. (B) Western blot analysis of THBS-1 protein expression levels in Hep-2 cells treated with 1 µmol/l 5-aza-dC at the indicated times. (C) Analysis of THBS-1 mRNA expression levels using reverse transcription-quantitative polymerase chain reaction in Hep-2 cells treated with 1 µmol/l 5-aza-dC at the indicated times. ^***P<0.001 vs. other 5-Aza-dC treatment time points. THBS-1, thrombospondin 1; Ctrl, control group; M, methylated bands; U, unmethylated bands.

Figure 4

Effect of 5-aza-dC on the viability and invasion of Hep-2 cells in vitro. (A) Cell viability was measured with Cell Counting Kit-8 following the treatment of Hep-2 cells with 5-aza-dC at different concentrations (0, 0.1, 1 and 5 µmol/l) for 24, 48, 72 and 96 h. (B) The number of cells that had invaded at the 24 h time point. The values represent the mean ± standard deviation. (C) Invasive ability of Hep-2 cells was determined using the Transwell assay following treatment with 5-aza-dC. Representative images of treated and untreated cells are presented (magnification, ×200). ^**P<0.01, ^***P<0.001 vs. the control group 5-Aza-dC, 5-aza-2′-deoxy-cytidine; OD, optical density; Ctrl, control.