脑小血管病血浆外泌体miRNA差异表达及其意义

Differentially expressed plasma exosomal miRNAs and their significance in patients with cerebral small vascular disease

目的探讨血浆外泌体miRNA在脑小血管病(CSVD)病人中的表达谱及临床应用价值。方法选择CVSD病人66例和健康体检者66例血液标本,分离血浆外泌体;随机选取其中16例CVSD病人和16例健康对照者血浆外泌体进行基因芯片检测构建差异表达的miRNA文库,对132例样本行PCR以检验测序结果。进行GO富集分析和KEGG分析。结果基因芯片测序显示,CVSD病人血浆外泌体中差异表达的miRNA共有38个,其中上调表达18个,下调表达20个。与健康对照者相比,miR-498、miR-320e、miR-6776、miR-455表达明显下调。实时荧光定量PCR验证结果与基因芯片结果一致。CVSD病人血浆外泌体miR-320e的表达水平较健康对照组显著下调,差异有统计学意义(t=4.661,P<0.001)。生物信息学分析表明,差异表达的miRNA的靶基因参与了细胞凋亡、阿尔茨海默病、FoxO信号通路、Wnt信号通路等生物学过程。结论CVSD病人血浆外泌体miRNA表达谱与健康对照者存在明显差异,miRNA可能通过靶基因及生物信号通路的调控作用参与了CVSD的发生与发展。

Objective To investigate the expression profile of plasma exosomal miRNAs and their application value in patients with cerebral small vascular disease(CSVD).Methods Blood samples were collected from 66 patients with CVSD and 66 healthy controls,and then plasma exosomes were isolated.Plasma exosomes were randomly selected from 16 patients with CVSD and 16 healthy controls,and gene microarray was used to construct a library of differentially expressed miRNAs.PCR was performed for all 132 samples to verify the results of sequencing.Gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes analysis were performed.Results Gene microarray showed a total of 38 differentially expressed miRNAs in plasma exosomes of CVSD patients,among which 18 were upregulated and 20 were downregulated.Compared with the healthy control group,the patients with CVSD had significantly downregulated miR-498,miR320e,miR-6776,and miR-455.The results of quantitative real-time PCR were consistent with gene microarray results.The patients with CVSD had a significantly downregulated expression level of plasma exosomal miR-320e compared with the healthy control group(t=4.661,P<0.001).The bioinformatics analysis showed that the target genes of differentially expressed miRNAs were involved in the biological processes including cell apoptosis,Alzheimer’s disease,the FoxO signaling pathway,and the Wnt signaling pathway.Conclusion There is a significant difference in the expression profile of plasma exosomal miRNAs between CVSD patients and healthy controls,and miRNAs may be involved in the development and progression of CVSD through the regulation of target genes and biological signaling pathways.