地西他滨上调唾液腺腺样囊性癌细胞系细胞hMLHl的表达

目的：通过检测地西他滨（decitabine）对体外培养人唾液腺腺样囊性癌（salivary adenoid cvstic carcinoma，SACC）细胞系细胞hMLHl的影响．探讨DNA甲基化转移酶抑制剂应用于SACC治疗的可行性及可能机制。方法：采用不同浓度的decitabine处理SACC细胞系SACC-83和SACC—LM细胞．观察细胞形态变化。选取5Ixmol／L的decitabine处理细胞后，分别使用甲基化特异性PCR、蛋白免疫印迹法、流式细胞技术检测用药前、后hMLHl基因启动子甲基化状况、hMLHl蛋白表达和细胞周期、凋亡的变化。应用SPSSl3．0软件包对数据进行独立样本t检验。结果：经decitabine处理后，SACC-83和SACC—LM细胞中hMLHl基因启动子甲基化水平降低，hMLHl蛋白表达水平分别升高1．582倍和1．977倍（P〈0．05）。用药后G0／G1期细胞比例显著减少．S期细胞比例显著增加（P〈0．05）．2种细胞系细胞晚期凋亡比例显著增加，差别有统计学意义（P〈O．05）。结论：DeCitabine可通过改变hMLHl基因启动子甲基化水平，上调蛋白表达；使SACC细胞阻滞于S期，Decitabine有可能作为SACC化疗药物或与顺铂联合使用，增强顺铂的效果．

Decitabine up-regulates the expression of hMLH1 gene in salivary adenoid cystic carcinoma cell lines

PURPOSE： To investigate the effect of decitabine, a methylation inhibitor, on hMLH1 gene expression in salivary adenoid cystic carcinoma （SACC）cell lines. METHODS： Methylation specific PCR （MSP） was used to test the methylation status in SACC cell lines before and after deeitabine treatment. And then the protein expression of hMLH1 was detected by Western blot. Flow cytometry was used to test the cell cycle and cell apoptosis before and after the treatment of decitabine. SPSS 13.0 software package was used for statistical analysis. RESULTS： MSP and Western blot showed that protein expression level of hMLH1 in SACC cells increased after treated by decitahine and correlated with the reversion of hypermethylation status. After treating with 5 ~Lmol/L decitabine for 48 h, cell cycles were blocked at S phase and some cells presented characteristic morphological changes of apoptosis. CONCLUSIONS： Deeitabine can up- regulate protein expression of hMLH1, which may be correlated with the reversion of hypermethylation status on these gene promoters in tumor cells. Supported by National Natural Science Foundation of China （81272976, 81072211） and the Ph.D. Programs Foundation of Ministry of Education （20120073110085）.