Table4_m6A mRNA Methylation Was Associated With Gene Expression and Lipid Metabolism in Liver of Broilers Under Lipopolysaccharide Stimulation.docx

Hepatic inflammation is always accompanied with abnormal lipid metabolism. Whether N6-methyladenosine (m6A) mRNA methylation affects irregular inflammatory lipid level is unclear. Here, the m6A modification patterns in chicken liver at the acute stage of LPS-stimulated inflammation and at the normal state were explored via m6A and RNA sequencing and bioinformatics analysis. A total of 7,815 m6A peaks distributed in 5,066 genes were identified in the normal chicken liver and were mostly located in the CDS, 3′UTR region, and around the stop codon. At 2 h after the LPS intraperitoneal injection, the m6A modification pattern changed and showed 1,200 different m6A peaks. The hyper- and hypo-m6A peaks were differentially located, with the former mostly located in the CDS region and the latter in the 3′UTR and in the region near the stop codon. The hyper- or hypo-methylated genes were enriched in different GO ontology and pathways. Co-analysis revealed a significantly positive relationship between the fold change of m6A methylation level and the relative fold change of mRNA expression. Moreover, computational prediction of protein–protein interaction (PPI) showed that genes with altered m6A methylation and mRNA expression levels were clustered in processes involved in lipid metabolism, immune response, DNA replication, and protein ubiquitination. CD18 and SREBP-1 were the two hub genes clustered in the immune process and lipid metabolism, respectively. Hub gene AGPAT2 was suggested to link the immune response and lipid metabolism clusters in the PPI network. This study presented the first m6A map of broiler chicken liver at the acute stage of LPS induced inflammation. The findings may shed lights on the possible mechanisms of m6A-mediated lipid metabolism disorder in inflammation.

肝脏炎症常伴随脂质代谢异常。目前尚不明确N6-甲基腺苷（N6-methyladenosine, m6A）mRNA甲基化是否会影响炎症状态下紊乱的脂质水平。本研究通过m6A测序、RNA测序及生物信息学分析，探究了脂多糖（lipopolysaccharide, LPS）诱导炎症急性期肉鸡肝脏与正常状态下的m6A修饰模式。在正常肉鸡肝脏中共鉴定到分布于5066个基因中的7815个m6A峰，这些峰主要定位于编码区（CDS）、3'非翻译区（3'UTR）以及终止密码子周边区域。在脂多糖腹腔注射2小时后，m6A修饰模式发生改变，出现了1200个差异m6A峰。高甲基化与低甲基化m6A峰的分布存在差异：前者主要定位于编码区，后者则多位于3'非翻译区及终止密码子附近区域。高/低甲基化基因分别富集于不同的基因本体（GO）条目及通路中。联合分析显示，m6A甲基化水平的变化倍数与mRNA表达的相对变化倍数呈显著正相关。此外，蛋白质相互作用（protein-protein interaction, PPI）预测分析显示，m6A甲基化及mRNA表达水平发生改变的基因聚集于脂质代谢、免疫应答、DNA复制及蛋白质泛素化等生物学过程。CD18与SREBP-1分别为免疫过程与脂质代谢过程中的核心枢纽基因。枢纽基因AGPAT2被证实可连接PPI网络中的免疫应答与脂质代谢两个基因簇。本研究首次绘制了脂多糖诱导炎症急性期的肉鸡肝脏m6A修饰图谱。本研究结果可为阐明炎症状态下m6A介导的脂质代谢紊乱潜在机制提供参考依据。