流感病毒感染A549细胞外泌体差异microRNA筛选及靶基因分析

Screening differentially expressed microRNA in exosomes from A549 cells infected with influenza virus and analyzing their target genes

目的:提取、鉴定甲型流感病毒(A/PR8/34,PR8)感染A549细胞外泌体,检测外泌体差异表达miRNA并进行靶基因预测、分析。方法:间接免疫荧光法检测PR8对A549细胞的感染率;超速离心法和免疫磁珠法提取、纯化PR8感染细胞外泌体(PR8-exos)及正常A549细胞外泌体(Con-exos);透射电镜观察外泌体形态和粒径大小;纳米粒径分析(NTA)检测外泌体粒径分布;Western blot检测外泌体标志蛋白;RNAseq检测外泌体差异表达miRNA;KEGG和GO富集分析差异miRNA靶基因。结果:100 TCID50的PR8对A549细胞感染率为100%;透射电镜观察PR8-exos及Con-exos均为杯托状双层膜小囊泡结构;PR8-exos和Con-exos粒径大小分布几乎一致,Con-exos浓度为8.3×10^(10) Particles/ml,PR8-exos浓度为1.6×10^(11) Particles/ml,PR8组外泌体浓度高于对照组;PR8-exos及Con-exos均检测出CD63、CD9和Hsp70蛋白表达,而未检测到calnexin;PR8-exos和Conexos差异表达miRNA共84个,PR8-exos差异表达miRNA对应的靶基因主要富集于细胞增殖、凋亡、病毒复制、感染、免疫等生物学过程。结论:流感病毒感染可刺激A549细胞外泌体分泌,外泌体差异表达miRNA参与调控的靶基因涉及细胞增殖和凋亡、固有免疫及炎症反应、干扰素相关信号通路。

Objective:To extract and identify exosomes from A549 cells infected with influenza A virus(A/PR8/34,PR8),to detect differentially expressed miRNA in exosomes,and to predict and analyze their target genes.Methods:Indirect immunofluorescence method was used to detect infective rate of PR8 on A549 cells;ultracentrifugation and immunomagnetic bead methods were used to extract and purify exosomes from A549 cell infected with PR8(PR8-exos)and exosomes from normal A549 cells(Con-exos);transmission electron microscope was used to observe morphology and particle size of exosomes;nanoparticle tracking analysis(NTA)was used to detect size distribution of exosomes;Western blot was used to detect protein markers of exosomes;RNAseq was used to detect differentially expressive of miRNA in exosomes;KEGG and GO enriched analysis was used to analyze target genes of differentially expressed miRNA.Results:Infective rate of PR8 with 100 TCID50 on A549 cells was 100%;PR8-exos and Con-exos were both cupholder-like bilayer membrane vesicles under transmission electron microscope;PR8-exos and Con-exos shared the same particle size distribution with concentration of PR8-exos as 1.6×10^(11) Particles/ml,which was higher compared to concentration of Con-exos as 8.3×10^(10) Particles/ml;CD63,CD9 and Hsp70 proteins were detected in both PR8-exos and Con-exos,while had no calnexin;a total of 84 differentially expressed miRNAs were detected in PR8-exos and Con-exos,with their target genes mainly enriched in biological processes such as cell proliferation,apoptosis,virus replication,infection,and immunity.Conclusion:Influenza virus infection can increase secretion of exosomes in A549 cells.Target genes of differentially expressed miRNAs are suggested to be involved in regulation of signaling pathways related to cell proliferation,apoptosis,innate immunity,inflammation,as well as interferon-related signaling pathways.