circRNAs were originally thought to be by-products of aberrant splicing with little functional potential[10]. Novel bioinformatic approaches coupled with biochemical enrichment strategies and deep sequencing have enabled comprehensive studies of circRNAs to be performed[9], and several studies have addressed the identification, characterization, and function of circRNAs during viral infection[32]. For example, human cytomegalovirus (HCMV) could cause significant changes in host circRNAs and it was found that circSP100 potentially bound to 10 HCMV-encoding proteins, which further influenced the process of HCMV infection[8]. The host circRNA-miRNA-mRNA network was significantly changed in human lung adenocarcinoma epithelial (Calu-3) cells infected with the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV), and circFNDC3B and circCNOT were also verified to reduce the MERS-CoV load by modulating various biological pathways, including the mitogen-activated protein kinase (MAPK) and ubiquitination pathways[36]. Nevertheless, the properties and potential roles of circRNAs during CV-A16 infection have not been thoroughly elucidated to date. In this study, we applied a next-generation sequencing technique to systematically analyze circRNA expression profiles in SH-SY5Y cells infected with CV-A16. In this way, thousands of circRNAs were identified from diverse genomic locations at different infectious times. Moreover, among these circRNAs, it was found that there were 1769 and 1192 showed differentially expressions in the CV-A16-12 h and CV-A16-24 h groups, respectively. While a small fraction of these circRNAs were intronic, nearly more than 90% of them originated from exonic. The large number of different circRNAs presented at different times indicated that the transcription of circRNAs was very active after CV-A16 infection. Functional circRNAs can directly affect host gene expression by regulating transcription or interfering with splicing, mainly due to that circRNAs are usually generated from host genes through backsplicing. Thus, the function of these dysregulated circRNAs was assessed by GO and KEGG enrichment analysis with their host genes. Furthermore, to further investigate the key circRNAs involved in the process of CV-A16 infection, we screened out only the common differentially expressed circRNAs during CV-A16 infection at different time points. The host genes that were related to these 554 overlapping dysregulated circRNAs participated in various biological processes, such as cellular process, metabolic process, biological regulation, localization, response to stimulus, signaling, developmental process, multicellular organismal process, biological adhesion, locomotion, immune system process, reproductive process, interspecies interaction between organisms, reproduction, biological phase, multi-organism process, and biomineralization, most of which were associated with initiation and progression of viral infection. Additionally, the KEGG annotation was also showed that these circRNAs might primarily regulate Gonadotropin-releasing hormone receptor pathway, EGF receptor signaling pathway, Wnt signaling pathway, FGF signaling pathway, Angiogenesis, Integrin signaling pathway, Inflammation mediated by chemokine and cytokine signaling pathway, PDGF signaling pathway, CCKR signaling map, and p53 pathway, which were involved in host regulatory mechanisms during virus infection. For instance, EGF receptor inhibitors have been found to inhibit hepatitis B virus (HBV) and hepatitis C virus (HCV) replication via downregulation of signal transducers and activators of transcription 3 (STAT3) phosphorylation[12]. Activation of the Wnt/β-catenin pathway increased influenza virus mRNA and virus production in in vitro in mouse lung epithelial E10 cells and mRNA expression of influenza virus genes in vivo in mouse lungs[25]. Thence, the above results prompted that the alterations of circRNAs induced by CV-A16 infection in SH-SY5Y cells might play important regulatory roles in CV-A16-infected SH-SY5Y cells by directly influencing host biological functions and pathways. Next, qRT-PCR methods were utilized to test and verify the reliability of RNA-seq data, which showed that the expression of selected random circRNAs was consistent with the sequencing results, although the fold-change was slightly different from the sequencing result.
As highly conserved endogenous RNAs, many circRNAs harbor abundant miRNA binding sites, indicating that they can sponge corresponding miRNAs and thus function as competing endogenous RNAs (ceRNAs) to regulate gene expression[30]. For instance, circRNA Cdr1as functioned as a ceRNA to promote hepatocellular carcinoma progression via sponging miR-1270 and enhancing the expression of AFP[29]. The circRNA_15698/miR-185/TGF-beta1 axis aggravated the extracellular matrix of diabetic nephropathy mesangial cells, which promoted diabetic nephropathy pathogenesis[16]. Thus, in order to investigate the potential functions of key circRNAs, we focused on the major significantly changed GO terms (i.e., “Immune system process”) and most correlated pathways (i.e., “Inflammation mediated by chemokine and cytokine signaling pathway”), and a circRNA-associated-ceRNA network was constructed. Most viruses are controlled satisfactorily by the immune system with limited damage to host tissues[31]. However, during the evolution of the viruses, they also can obtain its survival in the host cell through an immune escape mechanism[26]. Thus, exploring the “Immune system process” induced by CV-A16 infection might provide more clues for understanding its pathogenic mechanism. In addition, accumulating evidence has reported that inflammation is a major driving force in virus infection[3, 17], and the development of neurological symptoms caused by CV-A16 is closely associated with inflammation of the nervous system. Hence, the investigation of “Inflammation mediated by chemokine and cytokine signaling pathway” could help us to further excavate potential mechanisms and therapeutic agents in CV-A16 infection progression. In the circRNA-miRNA-mRNA triple network map, it was seen that miRNA that potentially bind to the circRNA and the most likely target genes to each miRNA were identified. Among these complicated networks, we paid particularly close attention to the circRNAs that may act as ceRNAs to regulate the expression of MMP2, which has previously been reported to participate in the regulation of viral infections. For example, MMP2 could mediate viral clearance during HBV infection by cleaving membrane-bound CD100 into soluble CD100 from T cells[34]. Dengue virus could directly infect macrovascular endothelial cells and result in overproduction of MMP-2, which might contribute to the pathogenesis of severe dengue infection[23]. In the current study, it was discovered 3 circRNAs-regulated axes on MMP2, namely hsa_circ_0004447/hsa-miR-942-5p/MMP2, hsa_circ_0078617/hsa-miR-6780b-5p/MMP2 and hsa_circ_0078617/hsa-miR-5196-5p/MMP2. Thence, it gave us a clear direction to study the specific mechanism of these circRNAs in the pathogenesis of CV-A16 infection.
In conclusion, in this study, we identified a population of circRNAs differentially expressed in CV-A16-treated and CV-A16-untreated SH-SY5Y cells by high-throughput sequencing analysis and verified the expression of 6 dysregulated circRNAs by qRT-PCR. Further analysis revealed that these common differentially expressed circRNAs in different groups may exert a crucial role in viral pathogenicity by participating in the regulation of viral infection-associated biological processes and signaling pathways. Moreover, the establishment of a ceRNA network further outlined the regulatory function of circRNAs that could potentially restrict or facilitate EV-A71 infection through regulating gene expression, especially 3 circRNAs regulatory axes involved in MMP2 regulation. Thus, the results of this study may be helpful for future studies investigating the molecular functions of circRNAs in viral pathogenesis and virus-host interactions of CV-A16 infection.