m^(6)A和miRNA协同调控北京鸭胚胎期骨骼肌发育的研究

Study on the Co-regulation of m^(6)A and miRNA on Skeletal Muscle Development of Peking Ducks in Embryonic Stage

[目的]试验旨在研究胚胎期miRNA和N6-甲基腺嘌呤(m^(6)A)在北京鸭胸肌发育中的调控作用,以期为了解北京鸭胸肌发育规律奠定基础。[方法]收集E13和E27两个阶段的北京鸭胚胎胸肌,利用甲基化RNA免疫共沉淀结合高通量测序(MeRIP-Seq)和小RNA高通量测序(miRNA-Seq)来鉴定在北京鸭胚胎胸肌细胞分化过程中可能存在的受m^(6)A调控的相关差异基因,再联合miRNA及其靶基因分析其功能。[结果]miRNA-Seq结果显示,差异显著miRNAs有181个,通过靶基因预测到11 176个靶标,包括m6A相关基因以及肌肉发育相关基因。MeRIP-Seq在E13和E27组分别检测到14 344和14 016个m^(6)A峰,分别映射到8 248和7 763个已知基因。E13与E27时期m6A共3 240个,映射到2 767个差异甲基化基因(differentially methylated genes, DMGs)。DMGs的GO和KEGG分析显示,DMGs主要富集于肌肉发育、脂肪沉积相关通路,同时也富集到很多和肌肉发育相关的基因,如MTPN、MYF6和MYF5。miRNA-Seq检测到5 556个差异表达基因(differentially expressed genes, DEGs),GO和KEGG通路分析结果表明,这些DEGs在Wnt信号通路、代谢途径、脂肪酸显著富集,提示DEGs可能参与了脂肪细胞分化和脂肪代谢过程。miRNA-Seq和m6A-Seq关联分析显示,共检测到1 517个差异甲基化和表达相关基因(differential expression and methylation genes, DEMGs),对这些DEMGs的GO和KEGG分析显示,DEMGs主要富集在肌肉发育相关通路中。[结论]m^(6)A甲基化修饰和miRNA对北京鸭骨骼肌发育和脂肪沉积有重要影响。

[Objective]The aim of this experiment was to study the regulation of miRNA and N6-methyladenosine(m^(6)A)in the development of breast muscle of Peking ducks at embryonic stage,in order to lay a foundation for understanding the development of breast muscle of Peking ducks.[Method]The breast muscles of duck embryo at E13 and E27 stages were collected.Methylated RNA immunoprecipitation with next generation sequencing(MeRIP-Seq)and microRNA sequcencing(miRNA-Seq)were used to identify potential differential genes regulated by m^(6)A in the differentiation process of breast muscle cells in Beijing duck embryos,and their functions were analyzed in combination with miRNA and its target genes.[Result]The miRNA-Seq results showed that there were 181 significantly different miRNAs,and a total of 11176 targets were predicted through target gene prediction,including m 6A related genes and muscle development related genes.14344 and 14016 m 6A peaks were detected by MeRIP-Seq in E13 and E27 groups,respectively,mapping to 8248 known genes and 7763 known genes.A total of 3240 m^(6)A were mapped to 2767 differentially methylated genes(DMGs)during E13 and E27 stages.GO and KEGG analysis showed that DMGs were mainly enriched in pathways related to muscle development and fat deposition,as well as many genes related to muscle development,such as MTPN,MYF 6 and MYF 5 genes.556 differentially expressed genes(DEGs)were detected by miRNA-Seq.GO and KEGG pathway analysis results indicated that these DEGs were significantly enriched in the Wnt signaling pathway,metabolic pathway,and fatty acid,suggesting that DEGs might be involved in adipocyte differentiation and fat metabolism processes.The association analysis between miRNA-Seq and m 6A-Seq showed that a total of 1517 differential expression and methylation genes(DEMGs)were detected.GO and KEGG analysis of these DEMGs showed that DEMGs were mainly enriched in muscle development related pathways.[Conclusion]m^(6)A methylation modification and miRNA had important effects on skeletal muscle development and fat deposition in Peking ducks.