Kruppel-like factor 4 expression in oral carcinoma cells and hypermethylation at the gene promoter

Background: Kruppel-like factor 4 (KLF4) is a transcription factor regulating proliferation-differentiation balance of epithelium, and down-regulated in less-differentiated and advanced oral carcinomas. Although the expression is inactivated by the promoter hypermethylation in malignant tumor cells, it remains unknown in oral carcinoma cells. Methods: Genomic DNA isolated from nine different oral carcinoma cell lines and a normal keratinocyte line was treated with sodium bisulfite, and methylation at KLF4 gene promoter was determined by PCR direct-sequence analysis. KLF4 expression in cells cultured with or without demethylation reagent was monitored by quantitative real-time PCR and immunoblot. Results: A 237-bp promoter region spanning - 718 and - 482 of KLF4 gene was hypermethylated in oral carcinoma cells that express KLF4 at a low level, but the methylation was infrequent in cells expressing KLF4 high amount. The downstream region from -481 to +192 was not methylated in any cell lines. Demethylation treatment of cells up-regulated the expression at mRNA and protein levels. Conclusion: This study demonstrated that hypermethylation at a narrow range of the promoter region downregulates KLF4 expression, and suggests that the loss of expression by the hypermethylation contributes to oral carcinoma progression.