基于RNA-seq的甲氨蝶呤处理sv129胚胎干细胞的转录组学分析

RNA-seq analysis of the transcriptome in sv129 embryonic stem cells treated with methotrexate

目的利用转录组测序(RNA sequencing,RNA-seq)技术探讨抗叶酸代谢药物甲氨蝶呤(methotrexate,MTX)处理的小鼠胚胎干细胞(mouse embryonic stem cells,mESC)基因转录表达的变化,并探讨其可能影响的生物学过程和关键信号通路。方法将mESC分为两组,其中对照组采用完全培养基培养,MTX组在此基础上加入0.12μmol/L MTX药物处理。MTX处理24 h分别收集细胞,提取总RNA进行RNA-seq,获得胚胎早期全基因转录组图谱并采用Deseq2软件筛选差异表达基因(differentially expressed genes,DEGs),采用生物信息学R软件包对MTX组与对照组进行差异基因的功能及通路分析。结果MTX组与对照组的转录组相比,共有365个DEGs,其中144个显著上调表达,221个显著下调。GO(Gene Ontology)功能富集分析显示,生物学过程(biological process,BP)分类集中在肌肉系统发育、肌肉收缩、肌纤维发育等肌肉系统发育过程,细胞组分(cellular component,CC)分类集中在细胞膜组分、肌原纤维、收缩纤维、肌丝、肌节等肌肉组分,分子功能(molecular function,MF)分类集中在跨膜转运蛋白结合活性及膜通道活性。KEGG(Kyoto Encyclopedia of Genes and Genomes)生物功能分析显示DEGs显著性富集的通路在钙信号通路、紧密连接。结论抗叶酸代谢药物甲氨蝶呤能够引起mESC基因组的转录改变,DEGs功能主要与肌肉发育以及肌肉纤维发育相关,同时DEGs富集的关键信号通路主要有肌肉发育相关的膜通道的钙离子信号通路和紧密连接。

Objective To investigate the effects of the methotrexate(MTX)on the gene expressions in mouse embryonic stem cells(mESC)and explore the underlying biological process and signaling pathways by RNA-sequencing(RNA-seq).Methods The mESC were divided into two groups:control group(complete medium)and MTX group(0.12μmol/L MTX).After MTX treatment for 24 h,the cells in each group were collected to extract the total RNA for RNA-seq.The differentially expressed genes(DEGs)of mESC were achieved by Deseq2 software.Furthermore,the function and pathway enrichment analysis of DEGs between control group and MTX group were also analyzed by R package.Results Compared with control group,there were 365 DEGs in MTX group,including 144 up-expressed genes and 221 down-expressed genes.Gene ontology(GO)functional enrichment analysis showed that the DEGs focused on muscle system process,muscle contraction,muscle fiber development,and other relevant muscle system processes in the biological process(BP);the cell membrane,myofibril,contractile fiber,myofilament,sarcomere and other muscle components in the cellular component(CC);and transmembrane transporter activity and membrane channel activity in molecular function(MF).KEGG biofunctional analysis showed that DEGs were significantly enriched in the calcium signaling pathways and tight junctions.Conclusion MTX can cause genome changes on the transcriptional level in the mESC genome.The functions of DEGs are primarily associated with muscle development and muscle fiber development.Meanwhile,DEGs mainly enrich the calcium signaling pathways and the tight junctions in membrane channels,which are related to muscle development.