小鼠神经母细胞瘤N2a细胞脑啡肽酶基因的表达与启动子区甲基化和组蛋白修饰相关

Expression of neprilysin gene is associated with methylation and histone modification on promoter in mouse neuroblastoma Neuro-2a cells

目的研究小鼠神经母细胞瘤Neuro-2a(N2a)细胞中脑啡肽酶(NEP)基因表达的表观调控机制,探讨DNA甲基化与组蛋白乙酰化之间的相互作用。方法体外培养N2a细胞,以3μmol/L、5μmol/L DNA甲基化酶抑制5-氮杂脱氧胞苷(5-Aza-dc)及(300、500、700)nmol/L组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)分别处理N2a细胞48 h和24 h。采用逆转录PCR(RT-PCR)、Western blot法分别检测NEP的mRNA、蛋白表达;亚硫酸氢盐测序聚合酶链反应(BSP)法检测NEP基因启动子区DNA的甲基化水平;染色质免疫共沉淀(ChIP)法检测NEP基因启动子区组蛋白H3的乙酰化水平。结果 5-Aza-dc和TSA均能剂量依赖地提高NEP基因的表达(P<0.01);5-Aza-dc可诱导NEP基因去甲基化(P<0.01);TSA可增高NEP组蛋白H3乙酰化水平(P<0.05),但不能明显改变NEP基因DNA的甲基化水平(P>0.05)。结论在小鼠神经母细胞瘤N2a细胞中NEP基因的表达受DNA甲基化及组蛋白乙酰化的调控,组蛋白乙酰化水平并不影响DNA甲基化状态。

Objective To investigate the epigenetic regulation of neprilysin （NEP） gene in mouse neuroblastorna Neuro-2a （N2a） cells and further determine the interaction between DNA methylation and histone acetylation. Methods N2a cells were treated with DNA methylation inhibitor, 5-aza-deoxycytidine （5-Aza-dc） at 3 and 5 ijmol/L for 48 hours and histone deacetylase inhibitor, trichostatin A （TSA） at 300,500 and 700 nmol/L for 24 hours. The expression of NEP after the treatment was evaluated by reverse transcription PCR（RT-PCR） and Western blotting. Bisulfite sequencing PCR （BSP） assay was utilized to detect the methylation status of NEP gene promoter regions. The level of acetylated histone H3 on NEP promoter was measured by chromatin immunoprecipitation （CHIP） assay. Results 5-Aza-dc induced the demethylation of NEP gene and significantly increased NEP expression in a dose-dependent manner （P 〈 0. 01）. TSA treatment significantly enhanced NEP expression by elevating the acetylation of histone H3 on NEP promoter （P〈0. 01, P〈0. 05）. However, methylation status of NEP promoter was not altered by TSA treatment （ P 〉 0. 05）. Conclusion The expression of NEP gene is regulated by DNA methylation and histone acetylation in N2a cells. Histone acetylation has no effect on DNA methylation.