皮肤恶性黑素瘤细胞株A375细胞RUNX3基因甲基化的研究

目的 研究皮肤恶性黑素瘤细胞株A375细胞中RUNX3基因启动子区CpG岛甲基化与其基因表达的关系，探讨RUNX3基因甲基化在皮肤恶性黑素瘤发病机制中的作用。方法 采用甲基化特异性PCR（MSP）检测A375细胞RUNX3基因启动子区CpG岛的甲基化状态，Western印迹检测A375细胞RUNX3蛋白表达水平。比较经不同浓度去甲基化药物５-杂氮胞苷（0、1、5、10、20 μmol/L）处理A375细胞后， RUNX3基因启动子区CpG岛的甲基化状态及蛋白表达水平的差异。结果 在A375中，处理前其RUNX3基因启动子区呈高甲基化状态，RUNX3蛋白表达缺失。经不同浓度５－杂氮胞苷处理细胞后，高甲基化的RUNX3启动子区发生不同程度去甲基化，表达缺失的 RUNX3蛋白不同程度恢复表达。结论 A375细胞株中RUNX3蛋白表达缺失可能与其启动子区的高甲基化有关； ５-杂氮胞苷能使A375细胞中的RUNX3基因发生去甲基化，从而重新激活因高甲基化而失活的基因，使RUNX3蛋白得以重新表达。

RUNX3 gene methylation in a cutaneous malignant melanoma cell line A375

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-azacyti-dine 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.