Transcriptome analysis and verification of differentially expressed genes before and after enterovirus D68 infection

Abstract

Objective To detect the alterations of the transcriptome in host cells infected by enterovirus D68 (EV-D68) and look for the genes involved in its infection so as to lay a foundation for the preliminary exploration of the infection mechanism. Methods The virus was inoculated into human rhabdomyosarcoma (RD) cells at a multiplicity of infection (MOI) of 0.01. After 24 h, RNA was extracted from the infected RD cells and control cells for transcriptome sequencing. Differentially expressed genes (DEGs) were screened and analyzed by using Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Eight DEGs, EGR1, c-fos, ZNF625, ARRDC3, c-jun, ASNS, CHAC1 and ADM2 were validated by RT-qPCR. Results A total of 140 genes with at least 2-fold changes at expression level were identified from the sequencing results, of which 71 were up-regulated and 69 were down-regulated. The results of GO functional enrichment analysis mainly involved the regulation and response to ions while the results of KEGG enrichment analysis mainly related to parathyroid hormone synthesis, secretion and action, IL-17 and GnRH signaling pathway, Toll-like receptor signaling pathway, and MAPK signaling pathway and so on. The RT-qPCR results showed that the trend of fold change of expression in the selected 8 genes was basically consistent with the sequencing results, and the expression levels of c-fos and c-jun were increased with the increase of virus MOI and infection duration. Conclusion By validating and using the functional and enrichment analysis, we infer that the mechanism of EV-D68 exacerbating asthma may be related to the up-regulation of c-fos, and MAPK signaling pathway may play a pro-inflammatory and antiviral role during EV-D68 infection.

Keywords

• ev-d68
• transcriptome sequencing
• differentially expressed genes