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背景与目的:缺氧对DNA错配修复系统(mismatch repair,MMR)活性的调控是肿瘤细胞遗传不稳定的重要原因,但其机制尚不完全清楚。本研究拟观察缺氧状态下人小细胞肺癌H446细胞DNA错配修复基因MLH1、MSH2的表达变化,初步探讨DNA甲基化在其中的作用。方法:应用RT-PCR、Western blot等方法检测H446细胞在缺氧状态下以及甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-CdR)处理后MLH1、MSH2基因的表达水平,同时,采用甲基化特异性PCR(MSP)方法检测MLH1、MSH2基因启动子CpG岛甲基化状态。结果:缺氧状态下,H446细胞MLH1、MSH2基因在转录和翻译水平均显著性降低。同时,随着缺氧时间延长,MLH1基因启动子逐渐由非甲基化状态、部分甲基化状态转变为完全甲基化状态,而MSH2基因启动子则直接由非甲基化状态转变为完全甲基化状态。甲基转移酶抑制剂5-Aza-CdR可使MLH1、MSH2基因表达水平有所恢复,但去除5-Aza-CdR后其表达再次下调。结论:启动子甲基化可能是缺氧诱导H446细胞显著性下调MLH1、MSH2基因表达的重要机制,甲基转移酶抑制剂5-Aza-CdR可恢复其表达。
BACKGROUND & AIM: The regulation of DNA mismatch repair (MMR) activity by hypoxia is an important reason for the genetic instability of tumor cells. However, its mechanism is not fully understood. This study was to observe the changes of DNA mismatch repair genes MLH1 and MSH2 in human small cell lung cancer H446 cells under hypoxia and to explore the role of DNA methylation in them. Methods: The expression of MLH1 and MSH2 genes in H446 cells under hypoxia and 5-Aza-CdR treatment with methyltransferase inhibitor 5-Aza-CdR were detected by RT-PCR and Western blot. Meanwhile, the methylation status of CpG islands of MLH1 and MSH2 gene promoters was detected by methylation-specific PCR (MSP). Results: Under hypoxia, MLH1 and MSH2 genes in H446 cells were significantly decreased at transcription and translation levels. At the same time, the promoter of MLH1 gene gradually changed from unmethylated state and partially methylated state to fully methylated state, while MSH2 gene promoter changed directly from unmethylated state to complete Methylation status. Methyltransferase inhibitor 5-Aza-CdR restored MLH1 and MSH2 gene expression, but its expression was down-regulated after 5-Aza-CdR was removed. CONCLUSION: Promoter methylation may be an important mechanism of hypoxia-induced down-regulation of MLH1 and MSH2 gene expression in H446 cells. Methyltransferase 5-Aza-CdR can restore its expression.