The prevalence of CV-B5 has been increasing every year, causing considerable threats to children under five years old. It also has been reported that adults infected with CV-B5 are at risk of developing severe clinical complications. Moreover, Central nervous system (CNS) pathology and neurological sequelae often occur after CV-B5 infection, which increases risk of mortality[15, 16]. In this study, the human neuroblastoma cell line, SH-SY5Y, was adopted for studying neurological damages of CV-B5 infection. CircRNA, a special class of endogenous non-coding RNA that plays an important role in the process of immune regulation has been shown to be involved in the development of a number of diseases. Therefore, studies of circRNA in virus-infected cells may provide important theoretical basis for viral disease treatment. In this study, RNA-seq was applied to investigate the circRNA expression profile of SH-SY5Y cells infected with CV-B5. The mechanisms of circRNAs identified to be involved in innate immune signaling pathway were further analyzed.
A total of 5,665 circRNAs identified to be present in SH-SY5Y cells are mainly derived from exons that are located on every chromosome except the Y chromosome. Only 3,167 circRNAs are found to be matched in the CircBase database, hence the rest are classified as newly discovered circRNAs. Moreover, 163 differentially expressed circRNAs were identified in CV-B5-infected cells. There were 26 differentially expressed circRNAs identified in both mock-infected cells and CV-B5-infected cells. We speculated that these 26 differentially expressed circRNAs may be cell-specific and unrelated to virus infection. An enrichment analysis showed that enriched GO-BP terms mainly focused on “metabolic process” and “protein modification” which is largely consistent with that of EV71 or CV-A16 virus infection virus infection. However, enrichment analysis of SH-SY5Y cells infected with EV71 or CA-16 indicates association with “gonadotropin-releasing hormone receptor pathway”, “Wnt signaling pathway”, “angiogenesis” and “p53 pathway”, which are not present in our analysis with CV-B5 infection[18, 19]. In our study, host genes that correspond to the differentially expressed circRNAs are mainly involved in “ubiquitin mediated proteolysis”, “tight junction” and the regulation of other signaling pathway, such as MAPK pathway. Ubiquitination, the most prevalent protein post-translational modification in cells, is known to play an important role in the dynamic regulation of host defense against pathogenic microbial infection[20, 21]. Ubiquitin often conjugates to lysine residues in substrate proteins, thereby inhibits the interaction of transcription factor with upper signal molecular. For example, STAT1 has linear ubiquitination which inhibits the binding of type-I interferon receptor IFNAR2, thereby restricting STAT1 phosphorylation, and resulting in type-I interferon signaling, in order to improve interferon (IFN) antiviral efficacy. In our study, the key junction molecules may undergo ubiquitination modification in order to regulate the antiviral immune response, although further validation is required. Tight junction may influence virus infection by blocking virus entry into the cells. MAPK signaling pathway has been shown to be involved in inflammatory response during IAV infection. Hence, our results collectively indicated that the altered expression of circRNAs may be the key regulators that participate in the infectious process of CV-B5.
An increasing number of studies have shown that circRNA function as works as miRNA sponges. CIRS-7, the first circRNA discovered, has been shown to play a role as a tumor-promoter by decreasing the regulation of miR-7 on central oncogenic factors via competitive interaction with miR-7. Subsequently, circRNA-miRNA interaction in viral infection were put forward. During the infection of Kaposis Sarcoma-Associated Herpesvirus, hsa_CIRC_0001400 has been shown to inhibit the expression of latency-associated nuclear antigen or replication and transcription activator in order to decrease virus infectivity through miR-K12-7-5p targeting. The same results have also been found in human cytomegalovirus and influenza A virus infection. Thus, it is necessary to establish differentially expressed circRNAs interaction network. In order to better understand the pathogenesis of CV-B5 infection that may facilitate the development of new therapeutic target, we focused on circRNAs interaction network in IFN pathway. The host innate immune system is the first line defense against viral invasion or replication. The association between IFN signaling pathway and the viral infection have widely documented, such as HBV, HSV and IAV[27, 28].
We found that hsa_circ_0008378 mainly affects the JAK-STAT/IFN signaling pathway and related ceRNA network. A total of 62 mRNA were identified to compete for 6 miRNAs including “hsa-miR-23a-3p/hsa_circ_0008378/TNFAIP3”, “hsa-miR-23a-3p/hsa_circ_0008378/DDX5”, “hsa-miR-23c/hsa_circ_0008378/BTLA” and “hsa-miR-23b-3p/hsa_circ_0008378/JAK1” from the IFN signaling pathway. TNFAIP3 (also known as A20), which is essential for maintaining immune homeostasis, is a key regulator of inflammatory, antiviral and apoptotic signaling pathways. DEAD-box polypeptide 5 (DDX5), also called p68, is an ATP-dependent RNA helicase. The roles of DDX5 in viral infection have been established. For instance, DDX5 has been shown as a host factor that exhibits antiviral activity during HBV and MYXV infection. Furthermore, Japanese encephalitis virus (JEV) and hepatitis C virus (HCV) have been found to hijack DDX5 to facilitate various steps of their replication cycles. B-and T-lymphocyte attenuator (BTLA), an immune-regulatory receptor, is known to promote HSV infection in host cells by forming an interactive network of CD160/BTLA/LIGHT/HVEM. Deubiquitinating enzyme HFMD virus structural protein VP3 has been shown to degrade Janus kinase 1 (JAK1) to inhibit IFN-γ signal transduction pathways. Also, we found that novel_circ_0014617 is enriched in RIG-I-like receptor signaling pathway, and that 33 mRNAs competitively bind to 2 miRNAs. Surprisingly, hsa-miR-138-5p was found to interact with lncRNA-GAS5, which has been shown to reverse cardiomyocyte injury by lower CYP11B2 expression. Additionally, we showed that “hsa-miR-138-5p/novel_circ_0014617/DNAJB6” and “hsa-miR-665/novel_circ_0014617/VPS4A” are enriched in the RIG-I signaling pathway. DNAJB6 has also shown to actively impact viral infection. Direct interaction in the DNAJB6 and prion protein during viral infection has been shown to favor nuclear localization of HIV-1 pre integration complex. In addition, the delivery of primase-UL70 into cell nuclei through DNAJB6 may promote the synthesis of viral DNA during HCMV infection. Although not all miRNA in the two circRNA networks selected in this study have binding with mRNA, they may be involved in CV-B5 infection, since they are the target molecules of circRNA. These miRNAs should be studied further, and the findings may provide new sights on CV-B5 infection.
In summary, our study revealed the characteristics and profiles of circRNAs in CV-B5-infected SH-SY5Y cells by RNA-seq. Differentially expressed circRNAs were identified and their potential functions were predicted by GO and KEGG. Enrichment analysis demonstrated that circRNAs associated with CV-B5 infection participate in cell metabolic process and immune response. Moreover, based on the six validated circRNAs, a circRNA/miRNA/mRNA network was constructed. Our analysis indicated that the circRNA played critical roles in the regulation of CV-B5 infection through IFN-I signaling pathway. Our results advance our current knowledge on the function of circRNAs in virus-host interactions. The findings in this study may also facilitate the identification of novel molecular targets for the prevention and treatment of CV-B5 infection.