感染LM小鼠巨噬细胞外泌体的转录组测序及差异表达miRNA特征分析  

Transcriptome sequencing and characterization of differentially expressed miRNAs in exosomes from mice macrophages infected by Listeria monocytogenes

作　　者：李能秀 焦健 左雨霏 马忠梅 常意行 孟庆玲[1] 才学鹏[2] 乔军[1] LI Neng-xiu;JIAO Jian;ZUO Yu-fei;MA Zhong-mei;CHANG Yi-hang;MENG Qing-ling;CAI Xue-peng;QIAO Jun(Department of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang 832003,China;China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]中国兽医药品监察所,北京100081

出　　处：《西南农业学报》2024年第7期1611-1621,共11页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(32160819,31360596);新疆维吾尔自治区研究生科研创新计划项目(XJ2021G104)。

摘　　要：[目的]分离和鉴定感染单核细胞增生李斯特菌(Listeria monocytogenes,LM)小鼠巨噬细胞外泌体(Exosomes,Exos)并分析其miRNA表达谱,为揭示巨噬细胞Exos miRNA在LM感染中的调控机制奠定研究基础。[方法]以未感染LM的巨噬细胞为对照组,感染LM的巨噬细胞为试验组,采用超速离心法分离提取巨噬细胞上清中的Exos,通过透射电镜、纳米颗粒追踪技术和Western blot对Exos形态、大小及表面标志分子特征进行鉴定。利用Illumina SE50测序平台对2组巨噬细胞Exos miRNA进行Small RNA-seq测序,筛选出显著差异表达的miRNA。使用Targetscan、miRDB和miRWalk数据库对差异表达miRNA靶基因进行预测,并对差异miRNA靶基因进行GO和KEGG-Pathway功能富集分析。利用Cytoscape软件绘制前20位Hub基因的miRNA-mRNA调控网络图。[结果]Exos直径为30~150 nm,具有典型的双层膜结构,Exos表面标志分子CD9、CD63和TSG101呈阳性表达。高通量测序结果显示,与对照组相比,试验组Exos中共筛选出9个显著差异表达的miRNA,其中显著下调基因8个(mmu-miR-7a-5p、mmu-miR-365-2-5p、mmu-miR-122-5p、mmu-miR-122b-3p、mmu-let-7i-5p、mmu-miR-151-3p、mmu-miR-182-5p和mmu-miR-1198-5p),显著上调基因1个(mmu-miR-192-5p),共预测miRNA调控靶基因1064个。GO富集分析结果显示,差异miRNA靶基因主要富集在轴突形成、细胞连接组装、肌动蛋白结合和金属离子跨膜转运等过程;KEGG分析显示,靶基因显著富集在cGMP-PKG信号通路和FcγR介导的吞噬作用通路。[结论]感染LM的小鼠巨噬细胞Exos miRNA表达谱发生了显著改变,其调控的潜在靶基因主要参与感染和免疫反应等信号通路。[Objective]The paper aimed to isolate and identify the Exosomes(Exos)of macrophages infected with Listeria monocytogenes(LM)in monocyte proliferation mice,and analyze their miRNA expression profile,laying a solid research foundation for revealing the regu-latory mechanism of macrophage Exos miRNA in LM infection.[Method]Macrophages infected with LM were used as the experimental group,while macrophages not infected with LM were used as the control group.Exos in the supernatant of macrophages were isolated and ex-tracted using ultracentrifugation.The morphology,size and surface marker characteristics of Exos were identified by transmission electron mi-croscopy,nanoparticle tracking technology and Western blot.Small RNA-seq sequencing of miRNA in macrophage Exos from the two groups was performed using the Illumina SE50 sequencing platform to screen for significantly differentially expressed miRNA.Targetscan,miRDB and miRWalk databases were used to predict the target genes of differentially expressed miRNA.GO and KEGG-Pathway functional enrich-ment analysis were conducted on the different miRNA target genes.The miRNA-mRNA regulatory network diagram of the top 20 Hub genes was drawn using Cytoscape software.[Result]The diameter of Exos ranged from 30 to 150 nm,with a typical double-layer membrane struc-ture,and the surface marker molecules CD9,CD63 and TSG101 were positively expressed.High-throughput sequencing results showed that compared to the control group,9 significantly diferentilly expressed miRNA were screened in the experimental group Exos,including 8 sig-nificantly down-regulated genes(mmu-miR-7a-5p,mmu-miR-365-2-5p,mmu-miR-122-5p,mmu-miR-122b-3p,mmu-let-7i-5p,mmu-miR-151-3p,mmu-miR-182-5p and mmu-miR-1198-5p)and 1 significantly up-regulated gene(mmu-miR-192-5p),with a total of 1064 predic-ted miRNA-regulated target genes.GO enrichment analysis results showed that the different miRNA target genes were mainly enriched in processes such as axon formation,cell junction assembly,actin binding and metal ion trans

关 键 词：单核细胞增生性李斯特菌 巨噬细胞 外泌体 MIRNA 靶基因 

分 类 号：S852.61[农业科学—基础兽医学]