基于RNA-seq技术研究埃可病毒30型感染RD细胞前后的差异表达基因

Exploration of Differentially Expressed Genes Before and After Infection with Echovirus 30 Based on RNA-sequencing

埃可病毒30型(Echovirus 30,E30)是一种全球传播的B组肠道病毒,常与无菌性脑膜炎等疾病暴发有关,分析E30在感染人横纹肌肉瘤(Human rhabdomyosarcoma,RD)细胞前后的差异表达基因有助于了解该病毒的复制周期以及宿主感染机制。本研究通过转录组测序技术探究E30感染RD细胞前后的基因表达谱变化,共检测到的1281个差异表达基因,其中包括730个下调基因和551个上调基因。基因本体论(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析表明,显著差异表达基因主要参与细胞受体信号通路的调节、炎症反应、免疫细胞活化、调控细胞生命周期等。利用荧光定量PCR(Realtime quantitative PCR,qPCR)对其中9个与炎症和免疫反应相关的差异表达基因进行验证,发现DEAD-box解旋酶3(DEAD-box RNA helicase 3,DDX3)表达上调,这与转录组学分析一致。利用RK-33(DDX3的小分子抑制剂)靶向抑制DDX3的表达,发现RK-33能够抑制E30的复制,并且qPCR结果显示在抑制DDX3的表达后,GTP酶激活蛋白结合蛋白1(GTPase-activating protein-binding protein1,G3BP1)和干扰素调节因子3(Interferon Regulatory Factor 3,IRF3)的表达也出现不同程度地降低。本研究的结果提示DDX3表达可能影响E30复制,这一发现为进一步探索E30在感染宿主过程中的分子机制奠定基础。

Echovirus 30(E30)is a globally transmitted species B enterovirus,which is often associated with aseptic meningitis and other disease outbreaks.Analysis of differential gene expression in E30 before and after infection with human rhabdomyosarcoma(RD)cells can aid understanding of the replication cycle of the virus and the infection mechanism in the host.We employed RNA-sequencing(RNA-seq)technology to explore changes in the gene-expression profile before and after E30 infection in RD cells.A total of 1281 differentially expressed genes(DEGs)were detected,730 of which had downregulated expression and 551 had upregulated expression.Analyses of enrichment of the function and signaling pathways of genes were done using the Gene Ontology(GO)database and Kyoto Encyclopedia of Genes and Genomes(KEGG)database,respectively.Genes with significantly different expression were involved mainly in regulation of“cell receptor signaling pathway”,“inflammatory response”,“immune cell activation”,and“cell life cycle”.Real-time quantitative polymerase chain reaction(qPCR)was used to identify nine DEGs related to inflammation and the immune response.Expression of DEAD-box RNA helicase 3(DDX3)was upregulated,which was consistent with the transcriptome analysis.RK-33(a small-molecule inhibitor of DDX3)could inhibit E30 replication.qPCR showed that expression of GTP enzyme activating protein binding protein-1 and interferon regulatory factor-3 decreased after inhibition of DDX3 expression.Results of this study suggest that DDX3 expression may affect E30 replication,which lays a foundation for further exploring the molecular mechanism of E30 infection.