高通量测序揭示EBV再激活过程中宿主细胞miRNA差异表达的动态变化  

High-throughput sequencing reveals the dynamic changes in the differential expression of cellular miRNAs during EBV reactivation

作　　者：李昊天 王慧[1] 王娇[1] 卢学新[1] 张杰琼 王明明 于东渤 李颖[1] 王世文[1] Li Haotian;Wang Hui;Wang Jiao;Lu Xuexin;Zhang Jieqiong;Wang Mingming;Yu Dongbo;Li Ying;Wang Shiwen(National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《中华实验和临床病毒学杂志》2025年第1期1-8,共8页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的明确EBV潜伏感染细胞中EBV再激活后宿主细胞miRNA表达谱的动态变化。方法使用佛波酯(TPA)诱导Raji细胞促使EBV激活进入裂解期,提取EBV激活后的细胞总RNA,构建sRNA文库,采用Illumina SE50平台进行高通量测序,通过生物信息学分析差异表达的miRNA,预测其潜在靶基因并进行功能富集分析;使用miRanda和RNAhybrid软件预测和筛选宿主细胞已知和未知miRNA中能够潜在靶向结合EBV基因组的miRNA。使用实时定量RT-qPCR验证差异表达的宿主细胞未知miRNA。结果通过高通量测序鉴定出宿主细胞miRNA共1301个,包括已知1189个、未知112个。其中差异表达的宿主细胞miRNA包括已知264个、未知13个。RNA二级结构预测结果显示未知miRNA均具有典型的pre-miRNA发夹结构。采用miRNA茎环法RT-qPCR对其中2个未知miRNA(Novel_miR_183和Novel_miR_242)进行验证,结果显示其在TPA激活组和未激活组间的表达差异与高通量测序结果中的差异无统计学意义(Novel_miR_183:F=1.407,P=0.3707;Novel_miR_242:F=1.277,P=0.3970)。宿主细胞差异表达的miRNA靶基因参与了多种重要的生物学过程和信号通路。1189个已知宿主细胞miRNA和108个未知miRNA可能靶向结合EBV基因组。结论EBV再激活导致Raji细胞中miRNA表达谱的显著变化,差异表达的miRNA可能靶向EBV基因组进而参与调控EBV的感染和复制过程,从而在EBV的感染、潜伏和再激活过程中发挥重要的调控作用。ObjectiveTo investigate the dynamic changes of cellular miRNA expression profiles in EBV latently infected Raji cells upon reactivation with Phorbol ester(TPA).MethodsTotal RNA was extracted using TRIzol reagent from Raji cells treated with TPA at different time points(0 h,24 h,48 h).Small RNA libraries were constructed and sequenced on an Illumina SE50 platform.Differentially expressed miRNAs were identified,and their target genes were predicted and functionally annotated.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were carried out through online tools.Additionally,miRanda and RNAhybrid software were used to predict cellular miRNAs targeting the EBV genome.Real-time RT-qPCR was employed to validate the expression levels of differentially expressed novel miRNAs.ResultsHigh-throughput sequencing identified 1301 celluar miRNAs,comprising 1189 known and 112 novel miRNAs.A total of 264 known differentially expressed cellular miRNAs and 13 novel miRNAs were identified through high-throughput miRNA sequencing.Secondary structure prediction revealed that the novel miRNAs exhibited typical pre-miRNA hairpin structures.Stem-loop quantitative real-time PCR(RT-qPCR)validation of Novel_miR_183 and Novel_miR_242 did not exhibit a statistically significant difference(F=1.407,P=0.3707 for Novel_miR_183;F=1.277,P=0.3970 for Novel_miR_242)between the TPA-stimulated and untreated groups.Target gene prediction analysis revealed that the differentially expressed cellular miRNAs were involved in various important biological processes and signaling pathways.Furthermore,1189 known cellular miRNAs and 108 novel miRNAs were predicted to target the EBV genome.ConclusionsTreatment of Raji cells with TPA stimulation successfully reactivated Raji cells and significantly altered their miRNA expression patterns.Differentially expressed miRNAs were identified,suggesting that these miRNAs probably play crucial roles in regulating EBV infection and replication by directly targeting the EBV genome.

关 键 词：EBV RAJI细胞 MIRNA 高通量测序 

分 类 号：R73[医药卫生—肿瘤]