目的 研究过量补充蛋氨酸（Met）对大鼠肝脏基因组甲基化状态的影响。方法 将24只Wistar大鼠分为对照组和蛋氨酸组,分别饲喂AIN-93M饲料和含有1.0% Met的AIN-93M饲料,时间为6 w。采用Arraystar Rat 4 × 180K RN4 RefSeq Promoter Microarray检测肝脏全基因组甲基化状况,NimbleScan v2.5软件处理芯片结果,DAVID软件进行GO和PATHWAY分析。分别采用荧光实时定量PCR法和ELISA法测定肝脏Ednra、Mrvi1和Gucy1A3的基因和蛋白表达水平。结果 补充Met后,在大鼠肝脏总共发现1092种差异甲基化启动子基因,涉及的基因与平滑肌收缩、血小板凝集、血压调节功能有关。信号通路分析显示。与血管平滑肌收缩相关的Mrvi1、Ednra和Gucy1A3变化显著,这3个基因启动子的DNA甲基化状态上调,而mRNA和蛋白表达减少。结论 过量补充Met影响大鼠肝脏基因组DNA甲基化状况;与血管平滑肌收缩相关的Mrvi1、Ednra和Gucy1A3基因是过量Met影响甲基化修饰的靶点。
Abstract
Objective To investigate the effects of excessive methionine (Met) on hepatic genome-wide methylated status in rats. Methods Twenty-four Wistar rats were allocated into two groups and were fed AIN-93M diet and 1.0% Met-supplemented AIN-93M diet, respectively, for 6 weeks. Hepatic samples were analyzed by Arraystar Rat 4 × 180K RN4 RefSeq Promoter Microarray for genome-wide methylated status. Microarray data was processed by NimbleScan v2.5 software. GO and PATHWAY analysis was performed by the Database for Annotation, Visualization and Integrated Discovery (DAVID) software. Hepatic mRNA and protein expressions of Ednra, Mrvi1 and Gucy1A3 were further assayed by quantitative PCR analysis and ELISA method, respectively. Results A total of 1,092 differentially methylated promoters were found in rat liver after 1.0% Met supplementation. Genes related to smooth muscle contraction, platelet aggregation, and regulation function of blood pressure were changed potentially in expression by excessive Met exposure. Six down-methylated and 13 up-methylated genes were found in several biological pathways after Met treatment, including Mrvi1, Ednra and Gucy1A3. The DNA methylation status of Mrvi1, Ednra and Gucy1A3 were up-regulated, and the mRNA and protein expressions were decreased after Met treatment. Conclusions Excessive methionine supplementation affects hepatic genomic DNA methylation status; Mrvi1, Ednra and Gucy1A3 which is closely associated with smooth muscle contraction, are targets of methylation modulation induced by excessive methionine exposure.
关键词
蛋氨酸 /
DNA甲基化 /
大鼠肝脏
Key words
methionine /
DNA methylation /
rat live
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基金
国家自然科学基金面上项目（No.81372988）
