The present study aimed to identify genes related to 5AZA-CdR in laryngeal squamous cell carcinoma (LSCC) and to investigate the role of S100A4 in the development and aggression of LSCC. Differentially expressed proteins were identified in Hep-2 cells treated with 5AZA-CdR by two-dimensional gel electrophoresis combined with MALDI-TOF-MS. mRNA, protein levels and DNA methylation status of S100A4 were assessed by RT-PCR, Western blotting and methylation-specific PCR, respectively. The invasiveness of Hep-2 cells transfected by siRNA S100A4 was determined by transwell migration assay. Protein profiles from Hep-2 cells treated with 5AZA-CdR were obtained, and several differentially expressed proteins such as S100 calcium-binding protein A4 (S100A4) were identified. Results of RT-PCR and Western blotting revealed that both mRNA and protein levels of S100A4 were significantly higher in the metastatic lymph nodes than those in paired adjacent normal laryngeal (PANL) or tumor tissues. The DNA methylation status displayed significant differences between the LSCC and the PANL tissues. The expression level of S100A4 decreased in Hep-2 cells undergoing RNA interference of S100A4. The number of cells which crossed the basement membrane filter was significantly lower in the RNAi S100A4 group when compared with the number in the control group. The abnormal expression of S100A4 identified in Hep-2 cells treated with an inhibitor of DNA methyltransferase appeared to result from the aberrant DNA methylation status of S100A4. The abnormal expression of S100A4 altered the invasiveness of LSCC.

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