缺氧对人小细胞肺癌H446细胞DNA错配修复基因MLH1、MSH2表达的影响及其机制探讨

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.

A study on regulation of DNA mismatch repair genes MLH1 and MSH2 in human SCLC cell line H446 under hypoxic condition

Background and purpose:Regulation of MMR activity under hypoxia may play an important role in genetic instability of cancer,but the mechanism is still unclear.We investigated the expression of DNA mismatch repair genes MLH1 and MSH2 in human SCLC cell line H446 under hypoxic condition and explore the role of promoter methylation of genes in hypoxia.Methods:RT-PCR and Western blot were applied to detect MLH1 and MSH2 expression in human SCLC cell line H446 at the mRNA and the protein level,respectively,under either hypoxic condition or after 5-Aza-CdR treatment.Meanwhile,methylation-specific PCR(MSP)was used to determine promoter methylation of MLH1 and MSH2.Results:The expression of MLH1 and MSH2 in H446 cells significantly decreased both at the mRNA and the protein level under hypoxic condition.5-Aza-CdR treatment led to the restoration of MLH1 and MSH2 expression,while,both MLH1 and MSH2 were down-regulated again after removing 5-Aza-CdR.Conclusions:The promoter methylation of MLH1 and MSH2 may play an important role in its defective expression in H446 cells under hypoxic condition.And 5-Aza-CdR could restore MLH1 and MSH2 expression.