High throughput sequencing of whole transcriptome and construct of ceRNA regulatory network in RD cells infected with enterovirus D68

Abstract

Background With the advancement of sequencing technologies, a plethora of noncoding RNA (ncRNA) species have been widely discovered, including microRNAs (miRNAs), circular RNAs (circRNAs), and long ncRNAs (lncRNAs). However, the mechanism of these non-coding RNAs in diseases caused by enterovirus d68 (EV-D68) remains unclear. The goal of this research was to identify significantly altered circRNAs, lncRNAs, miRNAs, and mRNAs pathways in RD cells infected with EV-D68, analyze their target relationships, demonstrate the competing endogenous RNA (ceRNA) regulatory network, and evaluate their biological functions. Methods The total RNAs were sequenced by high-throughput sequencing technology, and differentially expressed genes between control and infection groups were screened using bioinformatics method. We discovered the targeting relationship between three ncRNAs and mRNA using bioinformatics methods, and then built a ceRNA regulatory network centered on miRNA. The biological functions of differentially expressed mRNAs (DEmRNAs) were discovered through GO and KEGG enrichment analysis. Create a protein interaction network (PPI) to seek for hub mRNAs and learn more about protein–protein interactions. The relative expression was verified using RT-qPCR. The effects of Fos and ARRDC3 on virus replication were confirmed using RT-qPCR, virus titer (TCID50/ml), Western blotting. Results 375 lncRNAs (154 upregulated and 221 downregulated), 33 circRNAs (32 upregulated and 1 downregulated), 96 miRNAs (49 upregulated and 47 downregulated), and 239 mRNAs (135 upregulated and 104 downregulated) were identified as differently in infected group compare to no-infected group. A single lncRNA or circRNA can be connected with numerous miRNAs, which subsequently coregulate additional mRNAs, according to the ceRNA regulatory network. The majority of DEmRNAs were shown to be connected to DNA binding, transcription regulation by RNA polymerase II, transcription factor, MAPK signaling pathways, Hippo signal pathway, and apoptosis pathway, according to GO and KEGG pathway enrichment analysis. The hub mRNAs with EGR1, Fos and Jun as the core were screened through PPI interaction network. We preliminarily demonstrated that the Fos and ARRDC3 genes can suppress EV-D68 viral replication in order to further verify the results of full transcriptome sequencing. Conclusion The results of whole transcriptome analysis after EV-D68 infection of RD cells were first reported in this study, and for the first time, a ceRNA regulation network containing miRNA at its center was established for the first time. The Fos and ARRDC3 genes were found to hinder viral in RD cells. This study establishes a novel insight host response during EV-D68 infection and further investigated potential drug targets.

Long non-coding RNA SNHG9 regulates viral replication in rhabdomyosarcoma cells infected with enterovirus D68 via miR-150-5p/c-Fos axis

“…Their relative propensity for binding to diverse interaction complexes, for instance, contributes to the diversity of this activation ( O’Donnell et al, 2012 ). Importantly, it has been demonstrated that c-Fos is a target gene for miR-150-5p ( Wang et al, 2021 ), which was consistent with our previous findings ( Si et al, 2021 ).…”

“…Bowtie2 (v2.2.5) was used to align clean reads to the gene set, a database generated by the Beijing Genome Institute (BGI) in Shenzhen that includes known and new, coding and non-coding transcripts, and then RSEM (v1.2.12) was utilized to calculate gene expression levels. The original sequencing data used in the article, and the original data processing are all consistent with the source of the published articles in our laboratory (Si et al, 2021).…”

“…In a preliminary study in our laboratory, c-Fos was demonstrated to have an antiviral effect on RD cells infected with EV-D68 ( Si et al, 2021 ). Overexpression of c-Fos in EV-D68 infection resulted in a significant decrease in viral VP1 expression at both the RNA and protein levels, according to this study.…”