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目的:研究5-氮杂-2’-脱氧胞苷(5-Aza-CdR)在卵巢癌细胞系SKOV3中对核苷酸切除交叉修复互补基因1(excision repair cross complementation group 1,ERCC1)表达的影响及可能的机制。方法:设计特异性针对DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)基因的shRNA转染入人卵巢癌细胞系SKOV3细胞中,Western blotting检测SKOV3细胞DNMT1以及ERCC1的表达变化;利用不同浓度5-Aza-CdR于不同时间点处理卵巢癌SKOV3细胞,Western blotting检测DNMT1和ERCC1蛋白在处理前后的变化,利用亚硫酸氢钠法检测ERCC1基因启动子区域甲基化水平。结果:0.5、1.0、2.0、4.0μmol/L的5-Aza-CdR作用于SKOV3细胞后,DNMT1表达水平呈浓度依赖性降低,而ERCC1表达水平呈浓度依赖性升高;使用终浓度为1.0μmol/L的5-Aza-CdR处理SKOV3细胞12、24、36 h后,DNMT1表达水平呈时间依赖性降低,而ERCC1表达水平呈时间依赖性升高,亚硫酸氢钠法检测示药物处理前ERCC1启动子区域处于高甲基化水平,在用1.0μmol/L的5-Aza-CdR处理后,其启动子发生了去甲基化。结论:5-Aza-CdR通过DNMT1调控卵巢癌SKOV3细胞中ERCC1基因的甲基化及其表达水平。
AIM: To investigate the expression of 5-Aza-CdR (5-Aza-CdR) on ovarian cancer cell line SKOV3 in excision repair cross complementation group 1 (ERCC1) Impact and possible mechanisms. METHODS: shRNA specific to DNA methyltransferase 1 (DNMT1) gene was designed and transfected into human ovarian cancer cell line SKOV3. The expression of DNMT1 and ERCC1 in SKOV3 cells was detected by Western blotting. -Aza-CdR was used to treat SKOV3 cells at different time points. The changes of DNMT1 and ERCC1 protein were detected by Western blotting before and after treatment. The methylation level of ERCC1 promoter region was detected by sodium bisulfite assay. Results: After treatment with 0.5,1.0,2.0,4.0μmol / L 5-Aza-CdR, the expression of DNMT1 decreased in a concentration-dependent manner and the expression of ERCC1 increased in a concentration-dependent manner. The final concentration of 1.0μmol / L 5-Aza-CdR treatment of SKOV3 cells at 12,24,36 h, DNMT1 expression in a time-dependent manner, while the expression of ERCC1 in a time-dependent manner, sodium bisulfite test showed that before treatment ERCC1 The promoter region is hypermethylated and its promoter is demethylated after treatment with 1.0 μmol / L 5-Aza-CdR. Conclusion: 5-Aza-CdR regulates ERCC1 methylation and its expression in ovarian cancer SKOV3 cells through DNMT1.