非酒精性脂肪性肝病的异常m6A修饰

Abnormal m6A modification innon-alcoholic fattyliverdisease

目的:非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)严重影响人们的健康,近来研究发现6-甲基腺嘌呤(N6-methyladenosine,m6A)甲基化参与机体脂质代谢过程,但仍缺乏针对NAFLD的m6A修饰水平的研究。本研究探讨NAFLD患者肝组织中m6A修饰水平的变化。方法:采用液相色谱质谱(liquid chromatography-mass spectrometry,LC-MS)分析NAFLD患者肝组织中总m6A修饰水平的变化,甲基化RNA免疫共沉淀(methylated RNA immunoprecipitation,MeRIP)和微阵列(包含44122个mRNA和12496个lncRNA)分析整个转录组的m6A修饰水平的变化,进而通过基因本体(gene ontology,GO)功能富集分析、基因和基因组京都百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析对差异修饰的mRNA进行功能注释,最后随机抽取4个mRNA和4个lncRNA,通过MeRIP及实时反转录聚合酶链反应(real-time RT-PCR)验证微阵列结果。结果:与对照组相比,NAFLD组m6A总修饰水平明显下降,NAFLD组有176个差异修饰的mRNA和44个差异修饰的lncRNA。其中15个mRNA和7个lncRNA m6A修饰水平上调,而161个mRNA和37个lncRNA m6A修饰水平下调;GO功能富集分析和KEGG通路富集分析显示差异修饰的mRNA主要富集于能量代谢、转录调控、翻译调控等生物学过程。结论:与正常肝组织相比,NAFLD患者肝中m6A甲基化修饰发生了显著改变,可能在NAFLD的发生、发展过程中发挥重要作用。

Objective:Non-alcoholic fatty liver disease has seriously affected people’s health.Recent studies have found that N6-methyladenosine(m6A)methylation is involved in the lipid metabolism process of the body,but the study on the level of m6A modification in NAFLD is still not available.This study aims to explore the changes in the level of RNA m6A methylation modification in NAFLD liver tissues,and to provide experimental and theoretical basis for in-depth study on the role of RNA m6A methylation in the occurrence and development of NAFLD.Methods:Changes in the m6A level in NAFLD liver tissues were measured by liquid chromatography-mass spectrometry(LC-MS).Total RNA was extracted from liver tissues of NAFLD patients or normal control individuals and subjected to methylated RNA immunoprecipitation(MeRIP)with microarray analysis(including 44122 mRNAs and12496 lncRNAs)to determine the changes in m6A modification levels across the entire transcriptome.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were performed to annotate the differentially modified mRNAs.Finally,4 mRNAs and 4 lncRNAs were randomly selected to verify the microarray results by MeRIP and real-time transcription polymerase chain reaction.Results:A total of 176 mRNAs and 44 lncRNAs were found to be differentially m6Amodified in the NAFLD group compared with the control group.Among them,15 mRNAs and 7 lncRNAs were hypermethylated in NAFLD,while 161 mRNAs and 37 lncRNAs were hypomethylated in NAFLD.GO and pathway analysis showed that the differentially modified mRNAs were enriched mainly in biological processes such as carboxylic acid metabolism and transcriptional regulation.Conclusion:The m6A modification profile is changed in NAFLD liver tissues compared with normal liver tissues,which may functionally impact the pathophysiological progress in NAFLD.