孕期饮酒介导的组蛋白低甲基化对子代心脏发育基因过表达的影响

Effects of histone hypomethylation induced by alcohol during pregnancy on overexpression of cardiomyogenesis genes offspring mice

目的:探讨组蛋白甲基化修饰失衡在孕期酒精暴露致子代心脏发育相关基因表达异常中的作用及其机制。方法:选取健康昆明小鼠,于孕期0.5 d开始给予56%乙醇(5 mL/kg)灌胃,同时给予G9a-组蛋白甲基转移酶(histone methyltransferase, HMT)抑制剂BRD4770(1 mg/kg)灌胃,于胚胎14.5 d、胚胎16.5 d和新生0.5 d收集子代小鼠心脏。RT-qPCR检测心脏发育相关基因Gata4、Cx43和β-MHC的mRNA表达水平,比色法检测HMT活性,Western blot检测小鼠心肌组织组蛋白H3K9me3、G9a-HMT、Cx43及β-MHC的表达水平。结果:比色法结果表明,孕期酒精暴露的子代小鼠心肌组织中HMT活性与生理盐水对照组相比显著降低(P<0.05);Western blot结果表明酒精组小鼠心肌组织中G9a-HMT及组蛋白H3K9me3表达水平与生理盐水对照组相比显著降低(P<0.05);RT-qPCR结果显示Gata4、Cx43和β-MHC的mRNA表达水平在酒精组显著升高(P<0.05),且Western blot结果也表明酒精组Cx43和β-MHC蛋白水平显著高于生理盐水对照组(P<0.05)。同时,G9a-HMT特异性抑制剂BRD4770能够进一步降低小鼠心肌组织中组蛋白H3K9me3甲基化水平(P<0.05),且Gata4 mRNA表达水平及Cx43和β-MHC的转录和翻译水平在抑制剂干预组较酒精组显著升高(P<0.05)。结论:G9a-HMT介导的组蛋白甲基化修饰失衡可能参与了孕期酒精暴露所致的子代心脏发育相关基因表达异常。

AIM: To investigate the effects of histone methylation on the abnormal expression of cardiomyogenesis genes caused by alcohol during pregnancy and the regulatory mechanism, and to provide a new idea and intervention targets for preventing and curing congenital heart disease. METHODS: The alcohol(56%, 5 mL/kg) and G9 a-histone methyltransferases(HMT) inhibitor BRD4770(1 mg/kg) were given by gavage in Kunming mice during embryo(E) 0.5~14.5 d, and the hearts of the mice in E14.5, E16.5 and post neonatal 0.5 d(PND0.5) were collected. The mRNA expression of Gata4, Cx43 and β-MHC genes was detected by RT-qPCR. The activity of HMT was measured by colorimetry. Meanwhile, the protein expression of histone H3 K9 me3, G9 a-HMT, Cx43 and β-MHC was determined by Western blot. RESULTS: The results of colorimetry showed that the activity of HMT in the heart of the offspring mice treated with alcohol during pregnancy was decreased significantly compared with normal saline group(P<0.05), and Western blot data showed that the expression of G9 a-HMT and histone H3 K9 me3 were apparently decreased in the same samples(P<0.05). The mRNA expression levels of Gata4, Cx43 and β-MHC in alcohol group were apparently increased compared with normal saline group(P<0.05). Meanwhile, the protein levels of Cx43 and β-MHC were increased significantly in the same samples(P<0.05). However, BRD4770, a G9 a-HMT inhibitor, further attenuated the level of histone H3 K9 me3, and further upregulated the expression of Gata4, Cx43 and β-MHC in the heart of the the mice treated with alcohol(P<0.05). CONCLUSION: Histone methylation modification imbalance induced by G9 a-HMT may be involved in the abnormal expression of cardiomyogenesis genes in the heart of offspring mice caused by alcohol during pregnancy.