Intrauterine virus infection has caused significant economic loss for those engaged in animal husbandry. Double-stranded viral RNA is produced in the life cycle of most viruses and serves as a PAMP in the viral recognition pathway. Virus infection is a complex biological process that is regulated by coordinated genes, including coding genes and non-coding RNA. Although some lncRNAs and mRNAs have been proven to play a role in viral infection, there has been no comprehensive investigation. In this study, rabbits were selected as experimental models and poly(I:C) was used as the virus mimic. RNA-seq technology was used for the first time to comprehensively and systematically analyze lncRNAs and mRNAs in poly(I:C)-treated ESCs.
We detected 20,494 mRNAs and 10,927 lncRNAs in ESCs of rabbits. Among them, we found that many genes were involved in the development and regulation of uterine function, such as PTGS2, TGFβ1, RDH10, SLC5A1, SMAD2, GDF9, FSHβ. Retinol dehydrogenase 10 (RDH10) is a key enzyme for vitamin A metabolism and is essential for embryonic patterning, morphogenesis and survival [30]. In addition, TGF-β1 (transforming growth factor-beta 1) is abundantly expressed in the endometrium and secreted into the uterine fluid to regulate uterine function [31]. Growth and differentiation factor 9 (GDF9) is secreted by oocytes and plays a leading role in the control of ovarian function in reproduction [32]. The lncRNA, MSTRG.259847.2, cis-regulates its target gene, SMAD2, which is an important regulator of pituitary function and reproduction that interacts with GDF9 and FSHβ to affect FSH synthesis [33]. We also identified new mRNAs and new lncRNAs in large numbers, which may provide insights into their function during viral infection. We also found 1311 DE mRNAs and 291 DE lncRNAs before and after poly(I:C) induction, and the number of up-regulated genes was much larger than the number of down-regulated genes, indicating that more mRNAs and lncRNAs were activated after infection.
LncRNA not only regulates the expression of adjacent protein-coding genes by cis-regulation [34, 35], but also the expression of genes on other chromosomes by trans-regulation [36, 37]. Our results showed 26 cis- and 1297 trans-regulatory sites. The DE regulatory lncRNAs produced by virus invasion, the host cell antiviral immune response, and by apoptosis could be useful as disease biomarkers or as potential therapeutic targets [38]. Compared with the control group, we found that MSTRG.92782.1 (log2FC = 12.37) and MSTRG.101521.9 (log2FC = -10.26) were the most significantly up-regulated and down-regulated lncRNAs after poly(I:C) treatment. A total of 671 target genes of MSTRG.92782.1 were found and all of them were trans-regulated, five trans-regulated target genes of MSTRG.101521.9 were also identified, which may have functions in the infection process. Prostaglandins (PGs) are involved in reproductive processes, such as ovulation, fertilization, embryo development and early implantation [39]. PTGS2 is a rate-limiting enzyme in prostaglandin synthesis and a key enzyme regulating PG synthesis during corpus luteum dissolution and embryo implantation [40]. In our results, PTGS2 expression was significantly up-regulated by 62 different lncRNAs, indicating that lncRNAs may regulate the endometrial secretory function by promoting prostaglandin production after exposure to poly(I:C).
We also performed functional analysis of DE mRNAs and lncRNAs. GO analysis showed that the target genes were involved in many biological processes, such as the development of various tissues and organs, regulation of the MAPK cascade, regulation of the JNK cascade, the immune response, signal transduction and metabolism. KEGG pathway analysis showed that differential target genes of lncRNAs were significantly enriched in the TNF signaling pathway, the toll-like receptor signaling pathway, the NOD-like receptor signaling pathway, and the NF-κB signaling pathway. In this study, the cis- and trans-targeted genes of seven DE lncRNAs were used to predict their potential roles in the regulation of the immune response triggered by poly(I:C). Combined with DE genes related to viral infection, we constructed an interaction network involving seven DE lncRNAs and 78 trans-target genes, mainly involving PI3K-Akt, toll-like receptors, RIG-I-like receptor, NOD-like receptor, MAPK, NF-κB, and the Jak-STAT signaling pathway. This revealed the potential function of DE-lncRNAs and mRNAs in viral regulation and the host’s immune response.
We next explored the role of these seven lncRNAs and their corresponding target genes in uterine infection and the immune response. Viral proliferation in cells triggers immediate (innate) and delayed (adaptive) immune responses [41, 42]. In this study, many proven immune genes were identified, such as the cytokines, IL-1β and IL-6, and the chemokines, CCL5 and CCL2 [43]. The seven verified lncRNAs targeted several different cytokines and chemokines that promoted or inhibited the antiviral response. Continued expression of these molecules could lead to pathological effects [44, 45] as their expression pattern changed in response to infection. Cytokines mainly regulate the inflammatory response and increase the adhesion of neutrophils to vascular endothelium, resulting in an influx of polymorphonuclear (PMN) cells and macrophages into the uterus [46]. The delicate balance between pro-inflammatory cytokines and anti-inflammatory cytokines plays a key role in pathogen clearance [47, 48]; however, we did not detect any changes in expression of typical anti-inflammatory cytokines such as IL-4, IL-10 and IL-13. There is sufficient evidence to show a significant correlation between pro-inflammatory cytokines and the severity and persistence of uterine infections [49–51]. The body's immune response is maintained in balance, but virus invasion can easily break the balance. We hypothesized that changes in key immune-related molecules may be responsible for the development of uterine infections. The expressions of lncRNAs are regulated by time, space, developmental stage and multiple genes [52, 53]. This characteristic of lncRNAs coincides with the dynamic changes in the development of the immune system, the rapid response to external antigens or invading pathogens, and the preservation of immune homeostasis. Better control of the inflammatory response may be beneficial for resistance to viral infection, and lncRNA could be a target in this process.
IFN-α/β production after viral infection is one of the main innate responses to the virus and also can enhance the adaptive immune response to the virus. IFN-α and -β are pleiotropic cytokines, which can antagonize the virus by inducing the expression of antiviral proteins [54, 55], including IFN-stimulated gene 15 (ISG15), 2', 5'-oligoadenylate synthetase 1 (OAS1), and MxGTPase 1 (MX1) as the best characterized examples [56]. These proteins amplify antiviral signals, degrade viral RNA and block viral mRNA transcription respectively [57–59]. Our results showed that poly(I:C) significantly increased these antiviral proteins, which may be related to the host response to viral infection. In our interaction results, MSTRG.102664.8 was significantly up-regulated and its trans-target gene IFN-β was also up-regulated, indicating that MSTRG.102664.8 may play a role in the production of downstream antiviral factors through up-regulation of IFN-β expression. After poly(I:C) stimulation, ISG15 increased significantly and the corresponding MSTRG.102664.8, MSTRG.137189.4, MSTRG.39626.1, MSTRG.68469.1 increased significantly; but, MSTRG.153189.1 decreased significantly, indicating that ISG15 was regulated by multiple lncRNAs at the same time. LncRNAs play an important regulatory role in the fight between virus and host, involving the transcription of virus and host genes, stability and translation of mRNAs and the antiviral response of the host [60, 61].