显微镜下多血管炎患者血浆外泌体中差异表达的miRNAs及其功能分析

Analysis of differentially expressed miRNAs in plasma exosomes and their functions in patients with MPA

目的:探究显微镜下多血管炎(MPA)患者血浆外泌体中差异表达的miRNAs及其功能。方法:超速离心分离纯化MPA患者及健康志愿者血浆外泌体,透射电镜和Western blot法进行鉴定;miRNeasy Micro Kit提取外泌体总RNA;illumina HiSeqTM2500MiSeq高通量测序检测miRNAs表达谱;生物信息学分析2组差异表达的miRNAs并预测其靶基因及靶基因潜在功能。结果:超速离心获得的微囊泡直径为30100 nm,表达蛋白CD63和TSG101。高通量测序分析显示,2组显著差异表达miRNAs共32个,其中12个表达上调,20个表达下调。GO富集分析显示差异表达的miRNAs靶基因生物学过程方面主要富集于细胞过程,细胞组分方面主要富集于细胞内和胞质,分子功能方面主要富集于蛋白结合。KEGG富集分析显示差异表达的miRNAs可显著富集于代谢相关信号通路、MAPK信号通路及TLR信号通路等。结论:MPA患者血浆外泌体miRNAs表达谱与健康志愿者相比具有显著差异,推测差异表达的miRNAs在MPA代谢、炎症和免疫调节方面发挥作用。

Objective:To investigate differentially expressed miRNAs and their functions in plasma exosomes of patients with microscopic polyangiitis(MPA).Methods:Plasma exosomes from MPA patients and healthy volunteers were extracted and purified by ultracentrifugation,and identified by electron microscopy and Western blot. Exosomal RNAs were extracted by miRNeasy Micro Kit.Profiles of exosomal miRNAs were analyzed by illumina HiSeqTM2500/MiSeq high-throughput sequencing. Bioinformatics methods were used to analyze differentially expressed miRNAs of two groups and to predict target genes and potential functions.Results:Vesicles obtained from plasma by ultracentrifugation were indeed exosomes,30~100 nm in diameter and expressed CD63 and TSG101 proteins. High-throughput sequencing showed 32 significantly differentially expressed miRNAs in two groups,of which 12 were up-regulated and 20 were down-regulated. Gene ontology(GO)enrichment analysis showed that target genes of differentially expressed miRNAs in GO-biological process were mainly enriched in cellular processes;GO-cell components were mainly enriched in intracellular and cytoplasm;GO-molecular functions were mainly enriched in protein binding. KEGG enrichment analysis showed that target genes of differentially expressed miRNAs in plasma exosomes in MPA were mainly enriched in metabolic related signaling pathways,MAPK signaling pathway and TLR signaling pathway.Conclusion:Plasma exosomal miRNAs expression profile in MPA patients is significantly different from healthy controls,which speculated that differential expression of plasma exosomal miRNAs in MPA may play a role in disease metabolism,inflammation and immune regulation.