Abberant Methylation Of TSLC1, Survivin And Stathmin Promoter In Laryngeal Squamous Cell Carcinoma

AIM: To explore the relationship between the promoter methylation statuses (TSLC1, survivin and stathmin genes)and laryngeal squamous cell cancer.METHODS: (1) DNA Preparation: Genomic DNA was extracted from normal laryngeal mucosa,polyp of vocal cord and laryngeal squamous cell cancers by proteinase K digestion and the phenol-chloroform extraction procedure. (2) Bisulfite modification: A total of 1μg of genomic DNA, obtained from culture cells or tissues, was modified by sodium bisulfite to convert unmethylated cytosines to uracils. (3) Total RNA Extraction and RT-PCR: Extraction of RNA from culture cells or tissues was performed using TRIzo1 reagent according to the manufacturer's protocols. Isolated total RNA (2μg) was reverse-transcribed to cDNA. TSLC1 mRNA, survivin mRNA, and stathmin mRNA expression were determined by RT-PCR. (4) Methylation-Specific PCR (MSP): MSP distinguishes unmethylated from methylated alleles based on sequence changes produced after the bisulfite treatment of DNA, which converts only unmethylated cytosine to uracil, and subsequent PCR using primers designed for either methylated or unmethylated DNA. (5) Statistical analysis: The SPSS Statistical Package program 10.0 was used for all analyses. Associations between the variables were tested byχ2 test. Value of less than 0.05 (P<0.05) was deemed significant.RESULTS: (1) Among the 40 patients with laryngeal squamous cell cancer, hypermethylation of TSLC1 promoter was detected in 21 cases (52.5%). There was no significant diference in hypermethylation in relation to pathological grade and clinical staging and classification, but a highly significant diference was observed between patients with and without lymphnode metastasis (N0 and N1) (P<0.05). TSLC1 promoter hypermethylation was detected in human laryngeal squamous cell line Hep-2.TSLC1 mRNA was not expressed in all laryngeal squamous cell cancers having hypermethylation of TSLC1 promoter and Hep-2 cells,whereas it was expressed in normal laryngeal mucosa,polyp of vocal cord and laryngeal squamous cell cancers without hypermethylation.(2) Survivin was expressed in 24 of 40 (60%) cases of laryngeal squamous cell cancer tissues and Hep-2 cells.In contrast, survivin was not expressed in normal mucosa and polyp of vocal cord. Survivin promotor was unmethylated in both normal and neoplastic tissues. (3) Stathmin was expressed in 21 of 40(52.5%) cases of laryngeal squamous cell cancer tissues and 1 of 5 cases of polyp of vocal cord tissues.The expression of stathmin gene was not detected in normal laryngeal mucosa and Hep-2 cells. Stathmin promoter was equally hemi-methylated in cancer and non-cancer tissues.CONCLUSIONS: (1) Hypermethylation of TSLC1 promoter is associated with loss of its transcription in laryngeal squamous cell carcinoma.The high frequency hypermethylation of TSLC1 promoter illustrates its potential clinical application as tumor marker for diagnosis and prognosis. (2) No obvious relationship was found between survivin/stathmin gene promoter methylation pattern and their expression.