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Laboratory of Fish Molecular Immunology, College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, ChinaLaboratory of Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, ChinaKey Laboratory of Exploration and Utilization of Aquatic Genetic Resources (Shanghai Ocean University), Ministry of Education, Shanghai, ChinaNational Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai, China
Long noncoding RNAs (lncRNAs) are a diverse subset of RNA species of noncoding transcripts that are usually longer than 200 nt. However, the biological role and function of many lncRNAs have not been fully identified. It has been shown that one potential function of lncRNAs is to act as a precursor miRNA and promote the production of multiple miRNAs. However, the function of the miiuy croaker lncRNA MIR122HG has not been explored. In the present study, we show that this differentially expressed teleost fish lncRNA can act as the host gene of miR-122-5p, regulate its expression, and indirectly regulate the expression of potential inflammatory target protein transforming growth factor-β–activated kinase 1. We show that MIR122HG can negatively regulate the transforming growth factor-β–activated kinase 1–triggered NF-κB and interferon regulatory factor 3 signaling pathways and subsequently attenuate the innate immune response. In addition, MIR122HG can promote the replication of Siniperca chuatsi rhabdovirus and exacerbate the pathological effects caused by viral infection. We conclude that the study of lncRNA–miRNA–mRNA interaction through bioinformatics analysis or experimental-supported analysis can provide information for further elucidation of the functions of fish lncRNAs in innate immunity.
Pathogen infection of organisms induces a series of immune responses that naturally occur for the survival of the organism and the maintenance of immune homeostasis. In these processes, pattern recognition receptors (PRRs) are responsible for detecting pathogen-associated molecular patterns in the host immune system (
). PRRs mediate the activation of different downstream signaling pathways by recognizing the specific molecular characteristics of different pathogenic microorganisms. The activated signaling pathway induces immune response through the production of various inflammatory factors and type 1 interferons (IFN-1) to resist microbial infection or the entry of foreign objects. PRRs mainly include toll-like receptors (TLRs), retinoic acid–inducible gene I (RIG-I)–like receptors (RLRs), nucleotide-binding oligomerization domain–containing protein–like receptors, C-type lectins, and some DNA sensors (
). TLRs can recognize both extracellular and intracellular viruses, whereas RLRs are intracellular virus sensors, which are mainly responsible for identifying viral nucleic acid that invades the cytoplasm (
). When the virus infects the host, the virus-specific recognition receptor TLRs detects the pathogen signal and then recruits the toll–interleukin (IL)-1 receptor domain–containing adapter-inducing interferon-β adaptor protein. Toll–IL-1 receptor domain–containing adapter-inducing interferon-β interacts with tumor necrosis factor receptor–associated factor (TRAF) 6 and TRAF3 (
). Activated TRAF6 recruits the kinase receptor–interacting protein 1, which then interacts with the protein and activates the transforming growth factor-β–activated kinase 1 (TAK1) complex, activates NF- κB and mitogen-activated protein kinases (MAPKs), and induces inflammatory factors (
). TAK1 also plays an important role in the RLR pathway. After RIG-I recognizes the virus, crucial adaptor protein mitochondrial antiviral signaling protein is recruited, and then interferon regulatory factor 3 (IRF3) is activated by the IκB kinase complex, thus promoting the mass production of interferon-stimulating genes (ISGs) and IFN-1 (
). The TAK1 molecule is indispensable in these two pathways, indicating that TAK1 is essential in the immune pathway.
TAK1, a serine/threonine kinase, is an evolutionarily conserved member of the MAPK family. TAK1 is an important molecule both in TLR and RLR signaling pathways; it could be activated by various proinflammatory cytokines, and it mediates downstream signal transduction (
). In mammals, several regulatory molecules regulate the activity of the innate immune response by TAK1. For example, apoptosis signal–regulating kinase 1 inhibits IL-1-induced NF- κ B activity by blocking TRAF6–TAK1 interactions (
). Moreover, S6K1 interacts with TAK1, thereby inhibiting the association of TAK1-binding protein 1 and p38α with TAK1, and finally induces the activation of NF- κB and AP-1 by inhibiting the activation of IκB kinase and p38/Jun N-terminal kinase, respectively (
). LncRNA is widely distributed, and lncRNA is found in animals, plants, yeast, and other organisms. It is less conservative in sequence, and only approximately 12% of the lncRNA of humans can be found in other organisms (
). The function of lncRNA involves almost all biological processes of biological physiology and pathology. It can not only regulate cell proliferation, differentiation, metabolism and other physiological processes but also participate in the regulation of various pathological processes of the body. Several important functions of lncRNAs have been identified, including acting as miRNA sponges, binding to RNA-binding protein, and involving in protein translation (
), and it acts as a precursor miRNA to coordinate with mature miRNA to regulate the expression of targeted genes, which is the most classic case that lncRNA functions as a host gene to regulate miRNA. Moreover, lncRNA Gm2044 as the miR-202 host gene indirectly regulates Rbfox2 expression and modulates NCCIT cell proliferation, providing new clues for the pathogenesis and treatment of male infertility in humans (
). Although this regulatory mechanism of lncRNA has been very mature in humans and mice, the research of lncRNA in lower vertebrates such as teleost fish is still unclear.
miRNA is another important member of the ncRNA family and has attracted much attention because of its important regulatory role in the past several decades. miRNA mainly affects the expression of the entire gene through base complementary pairing with the seed sequence to the 3′-UTR of the target mRNA (
). Generally, miRNA has two modes of action, such as degrading the stability of mRNA and inhibiting the translation process of coding genes. In view of the interaction between miRNA and coding genes, the function of miRNA can be used to determine drug treatment targets for various diseases. The process of miRNA formation is well known, and the pre-miRNA undergoes a series of steps such as splicing in the nucleus and finally being transferred to the cytoplasm to obtain a mature miRNA (
). H19 is dependent on miR-675 to enhance the proliferation and invasion of AGS cells in gastric cancer, and the expression of miR-675 is positively correlated with the host gene H19 of miR-675 in patients with gastric cancer (
). The knockdown of the lncRNA H19/miR-675 axis regulates myocardial ischemic and reperfusion injury by targeting peroxisome proliferator–activated receptor alpha to protect the heart against myocardial ischemia/reperfusion injury (
). However, the study of miRNA derived from lncRNA in fish is still in a blind spot.
Generally, fish is an excellent biological model for studying the origin of the immune system. Here, we studied a new immune regulation mechanism of teleost fish after virus infection. Through high-throughput sequencing, we identified that the expression of lncRNA MIR122HG was significantly upregulated under viral stimulation. MIR122HG can regulate the production of miR-122-5p, similar to the function of pre-miR-122. We predicted that TAK1 is one of the potential targets of miR-122-5p and confirmed that miR-122-5p can inhibit TAK1 expression at different levels. Moreover, MIR122HG, which is the host gene of miR-122, can downregulate the activity of TAK1 by deriving miR-122-5p. In addition, TAK1 could negatively regulate the innate immune response induced by Siniperca chuatsi rhabdovirus (SCRV), thereby promoting the replication of SCRV. Our research provides information for the in-depth understanding of the relationship of lncRNA–miRNA–mRNA and reveals the important position of lncRNA in the immune response of fish.
Identification of MIR122HG
Recent studies have revealed the regulatory potential of many lncRNAs, such as participation in antiviral response. To investigate the function of lncRNA in teleost antiviral immune responses, we treated miiuy croaker with SCRV for 48 h and then performed RNA-Seq analysis by comparing the differential expression of lncRNA between SCRV untreated and treated groups. Approximately 897 differentially expressed lncRNAs have been found (
), including MIR122HG. To further verify the RNA-Seq results of the significant abundance of MIR122HG, we performed quantitative real-time PCR (qPCR) analysis of MIR122HG expression in miiuy croaker SCRV-treated spleen and Miichthys miiuy spleen cell (MSpC). The results showed that SCRV remarkably increases the expression of MIR122HG, both in spleen tissue (Fig. 1A) and MSpCs (Fig. 1B), suggesting that MIR122HG may be an immune-related ncRNA. We then evaluated the expression levels of MIR122HG in M. miiuy intestine cells (MICs), MSpCs, M. miiuy kidney cells, M. miiuy muscle cells, and M. miiuy brain cells (Fig. 1C). Among the aforementioned cell lines, MSpCs and MICs showed the highest and the lowest expression of MIR122HG, respectively. Therefore, MIR122HG is an immune-related lncRNA. Then, we used single-molecule full-length transcript sequencing, and the results showed that the length of MIR122HG was 945 bp, which is consistent with that of the Larimichthys crocea and Nibea diacanthus, located in chromosome 18 between genes ALPK2 and NEDD4L (Fig. 1D). Finally, the coding potential calculator computational algorithm was used to predict the coding ability of MIR122HG (Fig. 1E). To further verify that MIR122HG does not have the potential to encode protein, we constructed MIR122HG expression plasmid with FLAG tag (MIR122HG-FLAG). The experimental results showed that MIR122HG-FLAG plasmid did not express protein (Fig. 1F). Therefore, MIR122HG has very low coding ability, which supports that MIR122HG is an ncRNA.
MIR122HG is associated with the host innate antiviral immunity
To further investigate the biological functions of MIR122HG, we verified the differential upregulation of MIR122HG in vivo and conducted in vitro experiments. As shown in Figure 2A, the full-length expression plasmid of MIR122HG was constructed and named MIR122HG. To determine whether MIR122HG acts as an lncRNA to exert its function, we designed three siNAs (siRNA1, siRNA2, and siRNA3) of MIR122HG and found that siRNA1 had a better knockdown effect (∼40% reduction) than si-negative control (si-NC). siRNA1 was designed on the MIR122HG sequence, except the pre-miR-122 sequence. Then the siRNA1 of MIR122HG was used in all subsequent experiments and written as si-MIR122HG (Fig. 2B). Then, we investigated the effect of MIR122HG on the regulation of immune response through the overexpression of MIR122HG in MICs and knockdown of MIR122HG in MSpCs with SCRV stimulation, respectively. Considering the important role of IFN, ISGs, and inflammatory cytokines in the clearance of invading viruses, we investigated the role of MIR122HG in regulating the expression of IFN, ISGs, and inflammatory cytokines. As shown in Figure 2C, the overexpression of MIR122HG decreased the expression levels of tumor necrosis factor alpha, IL-8, IFN-2, and Mx1 in MICs under SCRV stimulation. By contrast, knockdown of MIR122HG significantly increased this indicated gene expression in MSpCs with SCRV stimulation (Fig. 2D). Moreover, 5-ethynyl-2′-deoxyuridine (EdU) assays were performed to detect cell proliferation rates for further exploring the role of MIR122HG in innate immunity. As shown in Figure 2, E and F, the overexpression of MIR122HG plasmid could decrease the cell proliferation rate, whereas transfection with si-MIR122HG could increase cell proliferation. Therefore, MIR122HG can inhibit cell proliferation. Overall, MIR122HG acts as a negative regulator to modulate the antiviral response and cell proliferation.
MIR122HG regulates the release of miR-122-5p
Many lncRNAs function as primary miRNA transcripts for miRNA to regulate mRNA expression (
). MIR122HG, an lncRNA with a dramatically increased expression upon SCRV stimulation, may participate in immune response in teleost fish. Through sequence alignment, we found that miR-122-5p is matched to MIR122HG precisely; thus, we further investigated the relationship between MIR122HG and miR-122-5p. To confirm the relationship between MIR122HG and miR-122-5p, we examined the expression of miR-122-5p in SCRV-infected spleen tissue and MICs at the same time. As shown in Figure 3, A and B, miR-122-5p levels could be dramatically increased in miiuy croaker SCRV-infected spleen tissue (Fig. 3A) and MICs (Fig. 3B).
We have demonstrated in Figure 2, A and B that MIR122HG and si-MIR122HG increased or inhibited the expression levels of MIR122HG, respectively. To further verify the relationship between MIR122HG and miR-122-5p, we detected the effect of gain and loss of MIR122HG on miR-122-5p expression. Figure 3C shows that increasing the levels of MIR122HG by transfecting MICs with the MIR122HG expression vector specifically induced the expression levels of miR-122-5p (Fig. 3C), suggesting that the processing of miR-122-5p from MIR122HG was increased by overexpressing MIR122HG. We also found that the expression of miR-122-5p was significantly reduced when MIR122HG was silenced, thus supporting that MIR122HG overexpression led to increased miR-122-5p levels. Therefore, MIR122HG may be the host gene of miR-122-5p. At the same time, we mutated all the sequences of the mature miR-122-5p on the MIR122HG overexpression plasmid to construct the mutant type (MT) of MIR122HG (MIR122HG-MT). Consequently, the plasmid no longer produced the mature miR-122-5p. We found that transfection of this MIR122HG-MT plasmid into MICs could not increase the level of miR-122-5p (Fig. 3C). This result confirmed that MIR122HG is the host gene of miR-122-5p and positively regulates miR-122-5p expression level. We also detected the expression of MIR122HG and miR-122-5p after transfection of pcDNA3.1, MIR122HG, MIR122HG-MT, NC, or si-MIR122HG in MICs for 24 h and treatment with SCRV infection for 24 h or treated poly (I:C) 18 h. As shown in Figure 3C, miR-122-5p and MIR122HG were highly expressed after transfection with MIR122HG in MICs under SCRV infection or poly (I:C) treatment. Importantly, upon SCRV infection or poly (I:C) treatment, the miR-122-5p level did not change when MIR122HG-MT was transfected into MICs. Therefore, regardless of viral infection, miR-122-5p is derived from MIR122HG.
TAK1 is a direct target of miR-122-5p
miRNAs affect various biological processes at the post-transcriptional level by targeting the 3′-UTR of mRNA; thus, we probed the potential target genes of miR-122-5p. Bioinformatics software programs were applied to seek the miR-122-5p targets, and we found that its potential target gene TAK1 is related to antiviral immunity. To obtain more direct evidence that miR-122-5p targets TAK1, we constructed the WT and MT of TAK1 3′-UTR reporter vector, as shown in Figure 4A. To detect the effect of miR-122-5p on the protein expression level of TAK1, we constructed the expression plasmid of TAK1 and transfected it with miR-122-5p, MIR122HG, and their respective controls into epithelioma papulosum cyprini (EPC) cells. As shown in Figure 4B, the results revealed that increased miR-122-5p expression led to a decrease in TAK1 protein. We also transfected miR-122-5p in different concentrations of the RNA or overexpressed MIR122HG in a concentration gradient manner in MICs. After 48 h, we detected by qRT–PCR, and the results showed that both miR-122-5p and MIR122HG reduced the expression level of TAK1 at the mRNA level in a dose-dependent manner (Fig. 4C). These results preliminarily demonstrated the inhibitory effect of miR-122-5p and MIR122HG expression on TAK1 at the post-transcriptional level.
To further clarify that this negative mechanism is realized through the complementation of miR-122-5p with seed sequences in the 3′UTR region, we performed dual luciferase reporting analysis. EPC cells were transfected with NC or miR-122-5p, along with TAK1 3′-UTR-WT or TAK1 3′-UTR-MT. After 24 h, the luciferase reporter assays showed that the overexpression of miR-122-5p led to a marked decrease of luciferase activity of the TAK1 3′-UTR-WT, whereas no change was observed in the luciferase activity of TAK1 3′-UTR-MT (Fig. 4D). In addition, we inserted the WT or a mutated form of TAK1 3′-UTR into mVenus-C1 vector and examined whether cotransfected with miR-122-5p could suppress the levels of GFP. As shown in Figure 4, E and F, miR-122-5p could significantly inhibit the levels of GFP, indicating the interaction between miR-122-5p and TAK1. To determine the function of MIR122HG on the luciferase activity of TAK1, we performed a similar experiment, and similar results were observed with MIR122HG (Fig. 4, G–I). Our results strongly indicate that TAK1 is the direct target of miR-122-5p.
MIR122HG exerts functions from miR-122-5p
Considering that miR-122-5p could regulate TAK1-mediated antiviral pathways, we investigated whether MIR122HG, as the primary precursor of miR-122-5p, can modulate the NF- κB and IRF3 signaling pathways through the functionality obtained from miR-122-5p. First, MICs were transfected with pcDNA3.1 or MIR122HG, and MSpCs were transfected with NC or si-MIR122HG and infected with SCRV for 6, 12, and 24 h. Then, qRT–PCR analysis was performed, and we found that the increased expression of TAK1 induced by SCRV was attenuated by MIR122HG, and the inhibitory effect was most obvious when SCRV was stimulated for 12 h (Fig. 5A, left panel). Knockdown of MIR122HG caused an increase in the expression level of TAK1 after intracellular infection of SCRV (Fig. 5A, right panel). Protein detection showed that MIR122HG inhibited the expression of TAK1 after SCRV infection, whereas MIR122HG silencing promoted the expression of TAK1 at the protein level (Fig. 6B). To determine whether MIR122HG could reduce TAK1-mediated signaling, we performed dual-luciferase reporter assay. TAK1 expression plasmid, pcDNA3.1, MIR122HG, MIR122HG-MT, pRL-TK vector, together with reporter genes, such as NF- κB, IL-8, IFN-2, and IRF3, were transfected into EPC cells. As expected, MIR122HG inhibited the luciferase reporter activity but not MIR122HG-MT (Fig. 6C). Then the concentration (Fig. 6D) and time gradient (Fig. 6E) assays were carried out. Results showed that MIR122HG could inhibit the luciferase activity in a dose-dependent manner.
Considering that miR-122-5p targets and regulates TAK1, we then tested whether miR-122-5p can regulate TAK1 upon SCRV infection. As shown in Figure 6A, overexpression of miR-122-5p led to a decrease in the expression levels of TAK1 in MSpCs treated with SCRV, whereas inhibition of miR-122-5p enhanced TAK1 expression in MICs. We then tested whether miR-122-5p regulates TAK1 expression at the protein level under SCRV treatment. Accordingly, we transfected with NC, miR-122-5p, miR-122-5p inhibitor (miR-122-5p-i), NC inhibitor, or miR-122-5p-i into MICs, respectively. Western blot analysis results showed that miR-122-5p could exert the inhibitory effect on TAK1, whereas miR-122-5p-i promoted TAK1 expression by counteracting miR-122-5p (Fig. 6B). Next, we investigated the influence of miR-122-5p on the TAK1-mediated signaling pathway. The results showed that TAK1 could activate NF- κB and IRF3 signaling, but the increased fluorescence activity decreased after transfection with miR-122-5p (Fig. 6C). Then, the concentration gradient of miR-122-5p was determined, and the results showed that the inhibitory effect was significantly increased with the increased transfection of miR-122-5p (Fig. 6D). Furthermore, the time gradient assays were constructed, and the luciferase activity was measured at indicated time points. The results showed that miR-122-5p inhibited TAK1-mediated NF- κB and IRF3 signaling pathways (Fig. 6E). Therefore, miR-122-5p plays a negative modulator in regulating signaling pathway.
MIR122HG enhanced SCRV replication
To explore the biological significance of MIR122HG upregulation in SCRV-induced host cells, we tested the effect of MIR122HG on SCRV replication in MICs. Previous studies on the involvement of miR-122-5p in antiviral immune response have reported that miR-122-5p promotes viral replication. Here, we investigated whether MIR122HG, a precursor of miR-122-5p, can exert a similar function. We examined the effect of MIR122HG on SCRV replication to explore the biological significance of MIR122HG in SCRV-induced host cells. Based on the detection of SCRV RNA level by qPCR, we found that MIR122HG knockdown significantly inhibited SCRV replication after 24 h of SCRV infection (Fig. 7A, left panel). Transfected MIR122HG plasmid significantly promoted SCRV replication but not the MIR122HG-MT plasmid after 24 h of SCRV infection (Fig. 7A, right panel). By measuring the SCRV 50% tissue culture infectious dose (TCID50) levels in the supernatant from the infected MSpCs and MICs, we found that MIR122HG knockdown significantly inhibited SCRV replication after 48 and 72 h of SCRV infection (Fig. 7B), and MIR122HG overexpression significantly promoted SCRV replication after 48 and 72 h of SCRV infection. Then, we investigated whether TAK1 can inhibit viral replication. Contrary to the effect of MIR122HG, the result shows that TAK1 can significantly inhibit SCRV replication (Fig. 7, C and D). Therefore, the host MIR122HG can promote SCRV replication.
MIR122HG regulating TAK1 is widely found in other teleost fish
To address the generality of our findings, we first examined the sequence alignment of MIR122HG from different lower vertebrate species. Interestingly, as shown in Figure 8A, mature miR-122-5p displayed high conservation in multiple fishes. The miR-122-5p-binding site in TAK1 3′-UTR also displayed a high conservation in other fish (Fig. 8, B and C, left panel). Then, we constructed the concentration gradient assays of LcrMIR122HG and NdiMIR122HG, and we found that LcrMIR122HG and NdiMIR122HG showed an obvious inhibition on TAK1 3′UTR luciferase activity in a dose-dependent manner (Fig. 8, B and C, middle). To obtain direct evidence that MIR122HG could regulate TAK1 3′-UTR across species, we generated luciferase report genes by cloning the TAK1 3′-UTR of L. crocea and N. diacanthus into pmir-GLO vector, within the devoid of miR-122-5p-binding site as an NC. Strikingly, LcrMIR122HG and NdiMIR122HG were sufficient to decrease luciferase activities when cotransfected with the WTs of L. crocea TAK1 3′-UTR and N. diacanthus TAK1 3′-UTR into EPC cells, respectively, but it showed no effect on the luciferase activity of cells transfected with their MTs (Fig. 8, B and C, right). In summary, these data indicate that miR-122-5p and MIR122HG could also target L. crocea and N. diacanthus TAK1 3′-UTR, miR-122-5p is highly conserved among in other teleost fish, and its precursor MIR122HG can also exert an inhibitory effect to modulate the expression of TAK1 genes in other species (Fig. 8D).
The study on the immunity of teleost fish, as an invertebrate to vertebrate transition, not only is an important part of vertebrate innate immunity but also is a key part of the evolution of innate immunity. Studies in the past decades have shown that teleost fish mainly rely on TLR3 and RIG-I to recognize RNA viruses, which invade the cells, recruit corresponding receptor proteins, and activate downstream signal cascades (
). TAK1 is essential for the response to the innate immune response mediated by these two PRRs. The activated TAK1 mediates the activation of NF- κB and IRF3 signaling pathways, leads to the production of inflammatory cytokines, chemokines, and type I IFN, and induces antiviral immune events (
). Although the host’s antiviral immunity is the most direct and effective way to resist virus invasion, excessive production of inflammatory factors and excessive induction of immune response can cause chronic inflammatory diseases or autoimmune diseases, which is detrimental to the body. Therefore, different levels of regulatory factors and mechanism networks are required to ensure the maintenance of the immune system’s homeostasis. Under normal circumstances, the maintenance of immune homeostasis is jointly regulated and supervised by many biological molecules, forming a complex immune regulatory network. In this article, we found that MIR122HG plays a negative regulatory role in teleost antiviral immunity. SCRV enhanced the expression of MIR122HG, and the upregulation of MIR122HG deteriorated inflammatory cytokines and ISGs. We further confirmed that MIR122HG can control the release of miR-122-5p and predicted TAK1 as a direct target of miR-122-5p. The assays demonstrated that both MIR122HG and miR-122-5p inhibited TAK1-mediated NF- κB and IRF3 signaling pathways by targeting TAK1, thus promoting SCRV replication (Fig. 8H). miR-217 can negatively regulate the antiviral immune response of miiuy croaker by targeting TAK1 (
). The regulation of TAK1 by miR-122-5p and miR-217 is both independent and coordinated because miRNA can regulate the translation of mRNA by combining its own seed sequence with the 3′UTR of mRNA, whereas the sequences of miR-122-5p and miR-217 are completely different. However, the regulation of TAK1 by the two miRNAs is coordinated with each other because the two miRNAs play a role through different sites on the 3′UTR of TAK1. The two miRNAs we found, miR-122-5p and miR-217, inhibit TAK1 after transcription. Because they both play an important regulatory role after SCRV infection, it shows that the two miRNAs can regulate TAK1 coordinately.
miRNA is a class of regulatory molecules that conserves sequence and has conserved function. The important regulatory functions of miRNAs are well known earlier than lncRNA. For example, miR-122 is dysregulated in various tumors, such as gastric, renal, and cervical cancer cells in human. miR-122-5p inhibits the proliferation, migration, and invasion of cancer cells by targeting LYN (an Src family tyrosine kinase) in gastric cancer cells (
). Recently, miR-122-5p inhibits charged multivesicular body protein 3 through MAPK signaling in breast cancer cells to promote aggressiveness and epithelial-to-mesenchymal transition in triple negative breast cancer (
miR-122-5p promotes aggression and epithelial-mesenchymal transition in triple-negative breast cancer by suppressing charged multivesicular body protein 3 through mitogen-activated protein kinase signaling.
). Although miR-122-5p plays a regulatory role in various cancer types, a few studies have revealed its effect on the innate immunity of viral infections. The present study revealed that fish miR-122-5p also can suppress the host’s antiviral immune response, indicating its important role in avoiding excessive immune responses.
Considering that the innate immunity of teleost fish is an important part of the innate immunity of vertebrates, the immune-related regulatory factors and regulatory mechanism of teleost fish should be studied. At present, the role of lncRNA as a fine-tuning regulator of different biological processes in humans and higher vertebrates has gradually been elucidated. The mechanism of competing endogenous RNA is one of the most studied hotspots, revealing that lncRNA slows down the inhibitory effect of mRNA by competitively binding miRNA. This mechanism was revealed in fish studies, and our previous research shows that antiviral-associated lncRNA (
) act as molecular sponges to bind miRNAs and slow down the adverse consequences caused by excessive miRNA suppression of the immune response. In addition, lncRNA can be used as the host gene of miRNA to regulate the production of miRNA to indirectly regulate mRNA. For example, in colorectal cancer cells, MIR100HG, which has a higher expression level, is inhibited by the gene GATA6, whereas miR-125b is derived from MIR100HG target GATA6, thereby lifting the inhibition of MIR100HG (
). Moreover, lncRNA MIR155HG showed cancer-promoting activity through miR-155-5p/-3p, and NSC141562 is an inhibitor molecule of the MIR155HG–miR-155 axis, which provides a new method for the treatment of glioma through a silent combination (
). The current view about the mechanism to form a mature miRNA from lncRNA is that this kind of lncRNA that can produce miRNA can mediate the termination of transcription through RNase III in mammals. Most of this lncRNA do not use the canonical cleavage-and-polyadenylation pathway but instead use microprocessor cleavage to terminate transcription, thus suggesting that splicing in lncRNA transcripts is not functionally important (
). In the present study, we found that fish lncRNA MIR122HG can produce mature miR-122-5p for the first time. We call it the MIR122HG–miR-122-5p axis, and this axis coordinately regulates innate immune response. We found that the MIR122HG–miR-122-5p axis showed high expression after viral infection and could negatively regulate the antiviral immunity mediated by SCRV. miR-122-5p targets TAK1 and inhibits the activity of TAK1. Moreover, MIR122HG downregulates the expression of TAK1. Therefore, MIR122HG, as an important negative regulator, controls the strength of immunity and plays an important role in fish antiviral immunity.
In the present study, we found a new lncRNA called MIR122HG in miiuy croaker and found that it can negatively regulate the antiviral immune response. Subsequently, we found that miR-122-5p can negatively regulate TAK1-triggered NF- κB and IRF3 signaling pathways. Further studies found that MIR122HG can produce miR-122-5p by acting as a precursor of miR-122-5p and then indirectly inhibit the TAK1-mediated antiviral immune response. Aside from mediating signaling, MIR122HG could be involved in SCRV-triggered antiviral immunity. In addition, the inflammatory cytokines and IFNI were suppressed by the enhancement of MIR122HG, thereby increasing SCRV replication. Our data provide new insights into the negatively feedback regulation of antiviral innate response in fish by lncRNA and the network enrichment on the host–virus interaction.
Animals and immune challenge
Miiuy croaker (∼50 g) was obtained from Zhoushan Fisheries Research Institute. Fish was acclimated in aerated seawater tanks at 25 °C for 6 weeks before experiments. Experimental procedures for SCRV infect was performed as described (
). All animal experimental procedures were performed in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, and the experimental protocols were approved by the Research Ethics Committee of Shanghai Ocean University (no. SHOU-DW-2018-047).
Cell culture and treatment
MICs, MSpC, M. miiuy kidney cells, M. miiuy muscle cells, and M. miiuy brain cells were cultured in L-15 medium (HyClone) supplemented with 15% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin at 26 °C (
). Human embryonic kidney 293 cells were cultured in Dulbecco's modified Eagle's medium, which contained the 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, and under humidified conditions with 5% CO2 at 37 °C (
To construct the TAK1 3′UTR reporter vector, the 3′UTR region of miiuy croaker TAK1 gene as well as L. crocea and N. diacanthus TAK1 3′UTR was amplified using PCR and cloned into pmir-GLO luciferase reporter vector (Promega). To construct MIR122HG expression plasmid, the full-length sequence of MIR122HG in aforementioned species was cloned into pcDNA3.1 vector or pcDNA3.1 vector with FLAG tag, respectively. The mutated forms with point mutations in the miR-122-5p-binding site were synthesized using Mut Express II Fast Mutagenesis Kit V2 with specific primers (Table S1). Meanwhile, miiuy croaker TAK1 3′UTR luciferase gene was inserted into the mVenus-C1 (Invitrogen), which included the sequence of enhanced GFP. Besides, the TAK1 expression plasmid was constructed as previously described (
). The predictions were ranked according to the predicted efficacy of targeting as calculated by applying the context and scores of the sites. Multiple sequence alignment of MIR122HG among the different species was conducted using DNAMAN8.0 (Lynnon Biosoft).
The miR-122-5p mimics are synthetic dsRNAs with stimulating naturally occurring mature miRNAs. The miR-122-5p mimics sequence was 5′-UGGAGUGUGACAAUGGUGUUUG-3’. The NC mimics was 5′-UUCUCCGAACGUGUCACGUTT-3’. miRNA inhibitors are synthetic ssRNAs that sequester intracellular miRNAs and block their activity in the RNA-interfering pathway. The miR-122-5p-i sequence was 5′-CAAACACCAUUGUCACACUCCA-3’. The NC inhibitor was 5′-CAGUACUUUUGUGUAGUACAA-3’. And the MIR122H G siRNA was synthesized and purified from GenePharma. Sequences are as follows: si-MIR122HG-1: 5′-GGUAUGAAU GAAUGAAUCUTT-3’; si-MIR122HG-2: 5′-GUCAUUUG UUU GUAUAUGUTT-3’; and si-MIR122HG-3: 5′-CUGUA UGUAUAGAUAAGAATT-3’.
Transient transfection of cells with miRNA mimic, miRNA inhibitor, or siRNA was performed in 24-well plates using Lipofectamine RNAiMAX (Invitrogen), and cells were transfected with DNA plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. For functional analyses, the overexpression plasmid (500 ng per well) or control vector (500 ng per well) and miRNA mimics (100 nM), miRNA inhibitor (100 nM), or siRNA (100 nM) were transfected into cells in culture medium and then harvested for further detection. For luciferase experiments, miRNA mimics (100 nM) or miRNA inhibitor (100 nM) and pmirGLO (500 ng per well) containing the WT or mutated plasmid of TAK1-3′UTR were transfected into cells.
Dual-luciferase reporter assays
To miRNA target identification, miR-122-5p mimics or NC, MIR122HG plasmid or pcDNA3.1 vector was transfected with TAK1 3′-UTR-WT or TAK1 3′-UTR-MT into EPC cells, respectively. At 48 h post-transfection, the activity of luciferase was detected using a dual-luciferase reporter assay system (Promega). To analyze the functional regulation of miR-122-5p, EPC cells were cotransfected with NF- κB, IL-8, IL-1β, or IRF3 luciferase reporter gene plasmids, TAK1 expression plasmid, and pRL-TK Renilla luciferase plasmid, together with miR-122-5p mimics, NC, MIR122HG expression plasmid, or empty vector, respectively. Then, the cells were lysed for reporter activity. All the luciferase activity values were achieved against the Renilla luciferase control. Transfection of each construct was performed in triplicate in each assay. Ratios of Renilla luciferase readings to firefly luciferase readings were taken for each experiment, and triplicates were averaged.
RNA extraction and real-time PCR
Total RNA was isolated with Trizol Reagent (Invitrogen), and the complementary DNA was synthesized using the FastQuant RT Kit (Tiangen), which includes DNase treatment of RNA to eliminate genomic contamination. The expression patterns of each gene were performed by using SYBR Premix ExTaqTM (Takara). The small RNA was extracted by using miRcute miRNA Isolation Kit (Tiangen), and miRcute miRNA First-strand cDNA Synthesis Kit (Tiangen) was applied to reverse transcription of miRNAs. The expression analysis of miR-122-5p was executed by using the miRcute miRNA qPCR Detection Kit (Tiangen). Real-time PCR was performed in an Applied Biosystems QuantStudio 3 (Thermo Fisher Scientific). β-actin and 5.8S rRNA were employed as endogenous controls for mRNA/lncRNA and miRNA, respectively (
Cellular lysates were generated by using 1× SDS-PAGE loading buffer. Proteins were extracted from cells and measured with the BCA Protein Assay kit (Vazyme), then subjected to SDS-PAGE (10%) gel, and transferred to polyvinylidene difluoride (Millipore) membranes by semidry blotting (Trans Blot Turbo System; Bio-Rad). The membranes were blocked with 5% bovine serum albumin. Protein was blotted with different antibodies. The antibody against TAK1 was diluted at 1:500; anti-FLAG and antitubulin monoclonal antibody was diluted at 1:2000 (Sigma); and the anti-GFP monoclonal antibody was diluted at 1:1000 (Sigma). The results were the representative of three independent experiments. The immunoreactive proteins were detected by using WesternBright TMECL (Advansta). The digital imaging was performed with a cold charge-coupled device camera.
EdU cell proliferation assay
The EdU dye assays were performed with BeyoClick EdU cell Proliferation Kit following the manufacturer’s instructions (Beyotime). All the experiments were performed in triplicate.
Data are expressed as the mean ± SD from at least three independent triplicated experiments. Student’s t test was used to evaluate the data. The relative gene expression data were acquired using the 2−ΔΔCT method, and comparisons between groups were analyzed by one-way ANOVA followed by Duncan’s multiple comparison tests (
The authors declare that they have no conflicts of interest with the contents of this article.
This study was supported by the National Natural Science Foundation of China (grant no.: 31822057 ).
T. X. conceptualization; T. X. methodology; R. C., W. Z., and T. X. formal analysis; R. C., W. Z., and T. X. investigation; R. C., W. Z., G. L., Q. L., and T. X. resources; R. C., W. Z., and T. X. writing–original draft.
miR-122-5p promotes aggression and epithelial-mesenchymal transition in triple-negative breast cancer by suppressing charged multivesicular body protein 3 through mitogen-activated protein kinase signaling.