寨卡病毒感染人肝癌细胞HepG2后外泌体中差异miRNA分析

Characteristics of the exosomal microRNA expression profile of HepG2 cells infected with Zika virus

目的探索人肝癌细胞HepG2感染寨卡病毒(Zika virus,ZIKV)后的外泌体miRNA的表达差异,对其靶基因进行预测、分析。方法以感染复数0.5的ZIKV(ZJ03株)感染HepG2,以超速离心法提取、纯化感染2 d与未感染细胞的外泌体(Exo-ZIKV与Exo-NC)。以透射电镜观察外泌体形态,纳米粒径分析检测外泌体粒径分布,Western blot检测外泌体标志蛋白质等方法鉴定。提取纯化外泌体小RNA,进行miRNA测序与分析,对部分差异表达miRNA采用实时定量PCR验证。生物信息方法分析差异miRNA靶基因。结果在透射电镜下呈杯托状双层膜小囊泡结构;粒径大小分布几乎一致但Exo-ZIKV浓度(5.24×10^(8)颗粒/ml)较Exo-NC(3.06×10^(8) paritcle/ml)增多。Western blot检测出Hsp70、CD63和CD9蛋白表达,测序分析显示共20个差异表达miRNA,其中12个miRNA表达上调8个miRNA表达下调,上调miRNA多与抗病毒通路相关,差异表达miRNA对应的靶基因主要富集在嘧啶与嘌呤代谢等途径中。结论ZIKV感染能刺激HepG2细胞外泌体分泌增加,且引起外泌体miRNA表达变化,这为深入了解ZIKV感染导致肝细胞损伤机制提供新基础,为ZIKV治疗提供新的思路。

Objective To isolate and identify the exosomes from human hepatocellular carcinoma(HepG2)cells infected with Zika virus(ZKIV),and anaylize the profiles of exosomal miRNA in infectedand uninfected cells,predict and analyze their target genes.Methods ZIKV(ZJ03 strain)was used to HepG2 cells at a multiplicity of infection(MOI)of 0.5.After 2 days,ultracentrifugation method were used to extract and purify the exosomes from ZIKV infected(Exo-ZIKV)and uninfected HepG2 cells(Exo-NC).The morphology and particle size of exosomes were observed by the transmission electron microscope;the size and concentration of exosomes were determined by Nanoparticle tracking analysis(NTA).Western blot was used to detect protein markers of exosomes.Small RNA was isolated from exosome and analyzed by small RNA deep sequencing.The differentially expressed miRNAs were further detected by bioinformatics analysis.KEGG and GO enriched analysis was used to analyze the target genes of differentially expressed miRNA.Results Under the transmission electron microscopy,Exo-ZIKV and Exo-NC were spherical or cup-shaped bilayer membrane structures.Exo-ZIKV and Exo-NC shared the similar particle size distribution,wheara Exo-ZIKV displayed the increased concentration(24×10^(8) paritcle/ml)than that of Exo-NC(3.06×10^(8) paritcle/ml).Typical exosomal markers Hsp70,CD63 and CD9 were detected in both gourps.A total of 20 differentially expressed miRNAs(12 up-rugalated and 8 down-regulated miRNA)were detected.The target genes of differentially expressed miRNAs were suggested to be involved in the pyrimidine and purine metabolism pathways.Conclusions ZIKV infection could promote the secretion of exosomes in HepG2cells.Furthermore,ZIKV infection cause the change of exosomal miRNA expression profile of HepG2 Cells.This study provides a basis for further elucidation of the role of exosomal miRNA in the mechanisms of ZIKV infection caused hepatocyte injury and provide new ideas for the treatment of ZIKV.