Abstract:

Objective To investigate the relationship between the methylation of CpG island of RUNX3 gene promoter and its expression in a human cutaneous malignant melanoma cell line A375, and to assess the role of RUNX3 gene methylation in the pathogenesis of human cutaneous malignant melanoma. Methods Cultured A375 cells were treated with various concentrations （0, 1, 5, 10, 20 μmol/l） of 5-azacytidine for 24 or 72 hours followed by another 5 days of culture. Then, methylation-specific PCR （MSP） was performed to evaluate the methylation status of RUNX3 promoter region, and Western-blot analysis to detect the protein expression of RUNX3 in A375 cells. Results The RUNX3 gene promoter region was hypermethylated in untreated A375 cells, along with the absence of protein expression of RUNX3. However, after the treatment with 5-azacytidine, the promoter region of RUNX3 gene was demethylated partly, and the expression of RUNX3 protein was restored in A375 cells. Further, the expression intensity was directly correlated with the concentration of 5-azacytidine. Conclusions The promoter hypermethylation of RUNX3 gene may be related to the silencing of RUNX3 gene expression in A375 cells, whereas 5-azacytidine can cause the demethylation of RUNX3 gene, reactivate the gene which has been inactivated by the promoter hypermethylation, and finally induce the re-expression of RUNX3 protein.