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目的:观察NK细胞系NK-92MI细胞中KIR3DL1基因启动子区的甲基化模式及去甲基化和组蛋白乙酰化对基因表达的影响,探讨KIR3DL1基因的表达调控机制。方法:采用亚硫酸氢盐测序法检测NK-92MI细胞中KIR3DL1基因启动子区的甲基化状况,应用甲基化抑制剂5-氮胞苷和(或)组蛋白去乙酰化转移酶抑制剂曲古抑菌素A处理NK-92MI细胞以诱导CpG岛去甲基化和组蛋白乙酰化,观察启动子区CpG岛甲基化和组蛋白乙酰化与KIR3DL1基因表达的关系。结果:NK-92MI细胞中KIR3DL1基因启动子区高甲基化,CpG二核苷酸甲基化频率在70%~100%之间;应用终浓度为2.5μmol/L和5μmol/L的5-氮胞苷作用72h可以诱导NK-92MI细胞中KIR3DL1mRNA表达量分别增加66.6%和114.6%;单用终浓度为50nmol/L的曲古抑菌素A不能诱导NK-92MI细胞中KIR3DL1mRNA表达量增加;此外,曲古抑菌素A和5-氮胞苷联用与单用5-氮胞苷相比也没有协同作用。结论:NK-92MI细胞中KIR3DL1基因表达受启动子甲基化调控。
OBJECTIVE: To observe the methylation patterns of KIR3DL1 promoter region in NK cell line NK-92MI cells and the effect of demethylation and histone acetylation on the gene expression, and to explore the mechanism of KIR3DL1 gene expression. Methods: The methylation status of KIR3DL1 gene promoter region in NK-92MI cells was detected by bisulfite sequencing. Methylation inhibitor 5-azacytidine and / or histone deacetylase transferase inhibitor Trichostatin A treatment of NK-92MI cells to induce CpG island demethylation and histone acetylation CpG island promoter methylation and histone acetylation and KIR3DL1 gene expression relationship. Results: The promoter region of KIR3DL1 gene was highly methylated in NK-92MI cells and the methylation frequency of CpG dinucleotide was in the range of 70% -100%. The optimal concentration of 5-azacytidine was 2.5μmol / L and 5μmol / L Glucosidase 72h could induce the expression of KIR3DL1mRNA in NK-92MI cells increased by 66.6% and 114.6%, respectively. Trichostatin A alone could not induce the increase of KIR3DL1mRNA expression in NK-92MI cells. Trichostatin A and 5-azacytidine combined with 5-azacytidine alone also had no synergistic effect. Conclusion: KIR3DL1 gene expression in NK-92MI cells is regulated by promoter methylation.