胃癌细胞系中组蛋白H3-K9甲基化与错配修复基因hMLH1表达及DNA甲基化的关系

Histone H3 lysine 9 methylation is associated with the expression of hMLHI and DNA methylation in gastric cancer cells

目的探讨胃癌细胞系中组蛋白H3-K9甲基化与DNA甲基化及错配修复基因hMLH1表达的关系。方法应用去甲基化制剂5-氮杂-2′-脱氧胞苷（5-Aza-dC）作用于2个胃癌细胞系，MGC-803和BGC-823。应用染色质免疫沉淀（CHIP）、甲基化特异性聚合酶链反应（MSP）和逆转录聚合酶链反应（RT-PCR）分析药物作用前后hMLH1基因启动子区域组蛋白H3-K9甲基化、DNA甲基化和hMLH1基因表达。结果MGC-803细胞系中，沉默的hMLH1基因启动子区域表现为DNA甲基化和组蛋白H3-K9高甲基化，5-Aza-dC不但能使DNA发生去甲基化，使沉默的hMLH1基因重新表达，而且能降低沉默位点的H3-K9甲基化水平。BGC-823细胞不表达hMLH1基因，其DNA非甲基化，与MGC-803细胞相比较，其组蛋白H3-K9甲基化水平低。5-Aza-dC对BGC-823细胞中的基因表达、DNA甲基化和R3-K9甲基化没有影响。结论在胃癌中，hMLH1基因启动子区组蛋白H3-K9的甲基化与DNA甲基化及基因沉默相关，去甲基化制剂可调控DNA甲基化、基因表达和组蛋白H3-K9甲基化。

Objective To identify the association of his tone H3 lysine 9 （H3-K9） methylation with DNA methylation and the expression of the mismatch repair gene hMLH1 in human gastric cancer cells. Methods Gastric cancer cells of the lines BGC-823 and MGC-803 were cultured and treated with 5- Aza-2＇-deoxycytidine （5-Aza-dC）, a demethylation agent, for 72 hour. Chromatin immunoprecipitation （CHIP） assay was used to assess the status of histone H3 lysine 9 methylation in the promoter regions of hMLH1 gene. Methylation-specific PCR （MSP） was used to evaluate the effect of 5-Aza-dC on DNA methylation status. RT-PCR was used to examine the hMLH1 gene expression. Results In the MGC-803 cells, silenced hMLH1 gene was characterized by DNA methylation and histune H3-K9 hypermethylation; 5- Aza-dC demethylated the DNA and reduced the histone H3-K9 methylation at silenced loci and resulted in reactivation of hMLH1 gene therein. Contrary to the MGC-803 cells, BGC-823 cells expressed hMLH1 gene with DNA demethylation and histone H3-K9 hypomethytation; and 5-Aza-dC had no effects on the gene expression, DNA methylation, and histone H3-K9 methylation therein. Conclusion Hypermethylation of DNA in the promoter region is related to transcriptional silencing of hMLH1 gene. Histone H3-K9 methylation in different regions of the promoter studied correlates with DNA methylation status of hMLH1 gene in gastric cancer cells. Alteration of DNA methylation affects histone H3-K9 methylation. 5-Aza-dC can control hMLH1 expression, DNA methylation, and histone H3-K9 methylation in the promoter.