Inducible MicroRNA-132 Inhibits the Production of Inflammatory Cytokines by Targeting TRAF6, TAK1, and TAB1 in Teleost Fish.

The innate immune response is the first line of defense against pathogen infection. Eradication of pathogen infection requires appropriate immune and inflammatory responses, but excessive inflammation may cause inflammatory and autoimmune diseases. MicroRNAs (miRNAs) are a group of small noncoding RNAs, and accumulating evidence has shown that in mammals, they can act as negative regulators that participate in the regulation of inflammation and immune responses. However, the miRNA-mediated immune regulation networks in the inflammatory responses of lower vertebrates are largely unknown. In this study, we report an miRNA, miR-132, identified from miiuy croaker, that acts as a negative regulator in the host's bacterium-induced inflammatory response. We found that miR-132 expression was dramatically increased upon infection by the Gram-negative bacterium Vibrio harveyi and lipopolysaccharide (LPS). Inducible miR-132 inhibits the production of inflammatory cytokines by targeting tumor necrosis factor receptor-associated factor 6 (TRAF6), transforming growth factor-activated protein kinase 1 (TAK1), and TAK1 binding protein 1 (TAB1), thus avoiding an excessive inflammatory response. Furthermore, we demonstrate that miR-132 modulates the inflammatory response through a TRAF6-, TAK1-, and TAB1-mediated NF-κB signaling pathway. These results collectively reveal that miR-132 plays a negative regulatory role in the host antibacterial immune response, which will help to gain insight into the intricate network of host resistance to pathogen infection in lower vertebrates.

microRNA-20-1 and microRNA-101a Suppress the NF-κB-Mediated Inflammation Production by Targeting TRAF6 in Miiuy Croaker.

Upon recognition of pathogen components by pattern recognition receptors, cells could be activated to produce inflammatory cytokines and type I interferons. The inflammation is tightly modulated by the host to prevent inappropriate inflammatory responses. MicroRNAs (miRNAs) are noncoding small RNAs that can inhibit gene expression and participate in various biological functions, including maintaining a balanced immune response in the host. To maintain the balance of the immune response, these pathways are closely regulated by the host to prevent inappropriate reactions of the cells. However, in lower vertebrates, the miRNA-mediated inflammatory response regulatory networks remain largely unknown. Here, we report that two miRNAs, i.e., miR-20-1 and miR-101a, were identified as negative regulators in teleost inflammatory responses. Initially, we found that both miR-20-1 and miR-101a dramatically increased after lipopolysaccharide (LPS) stimulation and Vibrio harveyi infection. Upregulated miR-20-1 and miR-101a inhibited LPS-induced inflammatory cytokine production by targeting tumor necrosis factor receptor-associated factor 6 (TRAF6), thus avoiding excessive inflammation. Moreover, miR-20-1 and miR-101a regulate the inflammatory responses through the TRAF6-mediated NF-κB signaling pathway. Collectively, these data indicate that miR-20-1 and miR-101a act as negative regulators by regulating the TRAF6-mediated NF-κB signaling pathway and participate in host antibacterial immune responses, which will provide new insights into the intricate networks of the host-pathogen interactions in the lower vertebrates.

MicroRNA-103 and microRNA-190 negatively regulate NF-κB-mediated immune responses by targeting IL-1R1 in Miichthys miiuy.

Accumulating evidence has demonstrated that microRNAs (miRNAs) regulate various physiological and pathological processes at the transcriptional level, thus called novel regulators in immune response. In this study, we used bioinformatics and functional experiments to determine the role of miR-103 and miR-190 in the regulation of IL-1R1 gene involved in the immune and inflammatory responses in miiuy croakers. First, we predicted the target genes of miR-103 and miR-190 through bioinformatics and found that IL-1R1 is a direct target gene of miR-103 and miR-190. This was further confirmed by the dual-luciferase reporter assay that the over-expression of miR-103, miR-190 mimics and the pre-miR-103, pre-miR-190 plasmids inhibit the luciferase levels of the wild-type of IL-1R1 3'UTR. miR-103 and miR-190 inhibitors increase the luciferase levels of IL-1R1-3'UTR. Additionally, we found that miR-103 and miR-190 could negatively regulate the mRNA expression of IL-1R1. Importantly, we demonstrated that miR-103 and miR-190 significantly inhibit the NF-κB signaling pathway by targeting IL-1R1 upon LPS stimulation. Collectively, these results provide strong evidence for an important regulatory mechanism of miR-103 and miR-190 targeting the IL-1R1 gene, thereby preventing excessive inflammatory immune responses from causing autoimmunity.

PCNA negatively regulates MITA through the autophagy pathway in miiuy croaker (Miichthys miiuy).

Interferon-mediated innate immune response is the first line of defense against foreign pathogen infection. Overexpression of MITA can activate the expression of interferon and promote the innate immune response of the body to the virus. These innate immune responses are tightly controlled to prevent the host from over-immunizing itself. In this study, we reported that structurally highly conserved PCNA negatively regulates MITA. PCNA overexpression can promote MITA degradation and block the expression of interferon, while the autophagy inhibitor 3-MA significantly inhibits MITA degradation, indicating that PCNA can degrade MITA through the autophagy pathway. PCNA inhibits interferon production by targeting MITA and avoids excessive immune response. In summary, our results indicate that PCNA is involved in the immune response by degrading MITA through the autophagy pathway, which will provide new ideas for further studies on the regulatory mechanism of immune signaling pathways in lower vertebrates.

Tinospora Cordifolia and Arabinogalactan in combination modulates benzo(a)pyrene-induced genotoxicity during lung carcinogenesis.

The present study explored the effects of combination of Tinospora cordifolia and Arabinogalactan on surface membrane dynamics and programmed cell deaths in rat model of lung cancer. The rats were divided into different groups namely normal control, benzo(a)pyrene (BP) treated, BP + Tinospora cordifolia (TC)-treated, BP + Arabinogalactan (A)-treated and BP + TC + A-treated groups. Significant changes were observed in the membrane dynamics of rats treated with BP. The carcinogen treatment demonstrated a marked decrease in membrane microviscosity. Also, excimer/monomer ratio and fluidity parameters of BP treated rats showed significant rise. On the other hand, combination of Tinospora cordifolia and Arabinogalactan improvised surface membrane dynamics. Moreover, micronuclei formation along with protein expression of bcl-2 showed significant increase in the lungs of BP treated rats. The combined treatment of Tinospora cordifolia and Arabinogalactan moderated the micronuclei formation in BP treated rats. Also, the combined treatment regulated the protein expressions of bcl-2 in BP-treated rats. As a result, marked improvement was noticed in apoptosis of BP treated cells treated with combination treatment. This study concludes that the Tinospora cordifolia and Arabinogalactan in combination improve the surface membrane dynamics and apoptosis in BP-treated rats.

microRNA-148 is involved in NF-κB signaling pathway regulation after LPS stimulation by targeting IL-1ß in miiuy croaker.

The inflammatory response is a protective process to clear detrimental stimuli, constitutes the defense against infectious pathogens. Clearing pathogen infection requires appropriate immune and inflammatory response, but excessive inflammatory response can lead to uncontrolled inflammation, autoimmune disease, or pathogen transmission. Accumulating evidences show that miRNAs are important and multifunctional regulators of innate immunity and inflammation. However, in the inflammatory response of lower vertebrates, the miRNAs regulatory networks are largely unknown. In this study, a combination of bioinformatics and experimental techniques were used to investigate the functions of miR-148. IL-1ß is a hypothetical target gene of miR-148 predicted by bioinformatics. In addition, dual-luciferase reporter gene experiment was used to verify the targeting effect of miR-148 on IL-1ß-3'UTR. miR-148 inhibits IL-1ß expression in a dose-dependent manner at protein and mRNA levels. It is important that miR-148 participates in regulation of LPS-induced the NF-κB signaling pathway by inhibiting IL-1ß. These results will improve our understanding of the regulation of miRNAs in fish on the immune response.

Rhabdovirus-Inducible MicroRNA-210 Modulates Antiviral Innate Immune Response via Targeting STING/MITA in Fish.

Viral infection induces type I IFN production, which plays critical roles in orchestrating the antiviral defense by inducing direct antiviral activities. To establish a persistent infection, viruses have evolved numerous strategies to specifically interfere with IFN production or its downstream mediators, thereby evading the immune responses. MicroRNAs (miRNAs) are a family of small noncoding RNAs that posttranscriptionally regulate the expressions of specific target genes. Although accumulating evidence demonstrates that miRNAs play vital roles in regulating viral infection, miRNAs that target intracellular sensors and adaptors of innate immunity have not been fully uncovered. In this paper, we identify fish miR-210 as a robust regulator involved in regulating virus-host interactions. We found that rhabdovirus significantly upregulated the expression of fish miR-210. Inducible miR-210 modulates virus-triggered type I IFN and inflammatory cytokine production by targeting stimulator of IFN genes (STING), thereby promoting viral replication. Furthermore, we demonstrated that miR-210 regulates innate immune response through NF-κB, IFN regulatory factor 3, and JAK/STAT signaling pathways. The collective findings indicate that inducible miR-210 plays a regulatory role in virus-host interactions through STING-mediated singling pathway by targeting STING.

The inducible microRNA-203 in fish represses the inflammatory responses to Gram-negative bacteria by targeting IL-1 receptor-associated kinase 4.

Innate immune responses are the first defense against pathogenic invaders. Activation and termination of these immune responses are regulated by several mechanisms. MicroRNAs (miRNAs), a group of small non-coding RNAs, have been implicated in the regulation of a spectrum of both physiological and pathological conditions, including immune responses. Although the immune regulatory miRNA networks in higher vertebrates have been well described, regulation of these responses in fish species is poorly understood. In the present study, we investigated the role of the miRNA miR-203 involved in inflammatory responses in miiuy croaker (Miichthys miiuy). We found that the Gram-negative bacterium Vibrio anguillarum and lipopolysaccharide significantly up-regulated host miR-203 expression. The increased miR-203 expression suppressed the production of inflammatory cytokines and thereby prevented mounting of a full immune response. Mechanistically, we identified and validated IL-1 receptor-associated kinase 4 (IRAK4) as a target of miR-203. We observed that miR-203 post-transcriptionally controls IRAK4 expression and thereby inhibits the activation of nuclear factor κB (NF-κB) signaling. In summary, our findings reveal that miR-203 in fish is a critical suppressor of innate immune responses to bacterial infection by suppressing a feedback to IRAK4-NF-κB-mediated signaling.

Inducible MicroRNA-3570 Feedback Inhibits the RIG-I-Dependent Innate Immune Response to Rhabdovirus in Teleost Fish by Targeting MAVS/IPS-1.

Effectively recognizing invading viruses and subsequently inducing innate antiviral immunity are essential for host antiviral defense. Although these processes are closely regulated by the host to maintain immune balance, viruses have evolved the ability to downregulate or upregulate these processes for their survival. MicroRNAs (miRNAs) are a family of small noncoding RNAs that play vital roles in modulating host immune response. Accumulating evidence demonstrates that host miRNAs as mediators are involved in regulating viral replication and host antiviral immunity in mammals. However, the underlying regulatory mechanisms in fish species are still poorly understood. Here, we found that rhabdovirus infection significantly upregulated host miR-3570 expression in miiuy croaker macrophages. Induced miR-3570 negatively modulated RNA virus-triggered type I interferon (IFN) and antiviral gene production, thus facilitating viral replication. Furthermore, miR-3570 was found to target and posttranscriptionally downregulate mitochondrial antiviral signaling protein (MAVS), which functions as a platform for innate antiviral signal transduction. Moreover, we demonstrated that miR-3570 suppressed the expression of MAVS, thereby inhibiting MAVS-mediated NF-κB and IRF3 signaling. The collective results demonstrated a novel regulation mechanism of MAVS-mediated immunity during RNA viral infection by miRNA.IMPORTANCE RNA viral infection could upregulate host miR-3570 expression in miiuy croaker macrophages. Induced miR-3570 negatively modulates RNA virus-triggered type I IFN and antiviral gene production, thus facilitating viral replication. Remarkably, miR-3570 could target and inhibit MAVS expression, which thus modulates MAVS-mediated NF-κB and IRF3 signaling. The collective results of this study suggest a novel regulation mechanism of MAVS-mediated immunity during RNA viral infection by miR-3570. Thus, a novel mechanism for virus evasion in fish is proposed.

MicroRNA-3570 Modulates the NF-κB Pathway in Teleost Fish by Targeting MyD88.

The inflammatory response, a protective process to clear detrimental stimuli, constitutes the defense against infectious pathogens. However, excessive inflammation disrupts immune homeostasis, which may induce autoimmune and inflammatory diseases. In this study, we report that microRNA (miR)-3570 plays a negative role in the bacteria-induced inflammatory response of miiuy croaker. Upregulation of miR-3570 by Vibrio anguillarum and LPS inhibits LPS-induced inflammatory cytokine production, thus avoiding an excessive inflammation response. Evidence showed that miR-3570 targets MyD88 and posttranscriptionally downregulates its expression. Overexpression of miR-3570 in macrophages suppresses the expression of MyD88, as well as its downstream signaling of IL-1R-associated kinases 1 and 4 and TNFR-associated factor 6. These results suggest that miR-3570 plays a regulatory in the bacteria-induced inflammatory response through the MyD88-mediated NF-κB signaling pathway by targeting MyD88.

Inducible microRNA-214 contributes to the suppression of NF-κB-mediated inflammatory response via targeting myd88 gene in fish.

Upon recognition of bacterial pathogens by pattern recognition receptors, cells are activated to produce pro-inflammatory cytokines and type I IFN by multiple signaling pathways. Every step of the process must be precisely regulated to prevent dysregulation. MicroRNAs (miRNAs) have been shown to be important regulators with profound effects on inflammatory response. Nevertheless, the miRNA-mediated regulatory mechanism remains unclear in fish species. Here, we addressed the role of miiuy croaker miR-214 in the bacteria triggered inflammatory response. miR-214 could significantly be up-regulated by Vibro harveyi and LPS stimulation. Up-regulating miR-214 subsequently inhibits the production of inflammatory cytokines by targeting myd88 to avoid excessive inflammation. Moreover, the negative regulatory mechanism of miR-214 has been demonstrated to be via the myd88-mediated NF-κB pathway. This is the first to focus on miR-214 acting as the negative regulator involved in the bacteria-triggered inflammatory response and thus may provide knowledge on the host-cell regulator responses to microbial infection.

MicroRNA-216a Inhibits NF-κB-Mediated Inflammatory Cytokine Production in Teleost Fish by Modulating p65.

Inflammation is the host self-protection mechanism to eliminate pathogen invasion. The excessive inflammatory response can result in uncontrolled inflammation, autoimmune diseases, or pathogen dissemination. Recent studies have widely shown that microRNAs (miRNAs) contribute to the regulation of inflammation in mammals by repressing gene expression at the posttranscriptional level. However, the miRNA-mediated mechanism in the inflammatory response in fish remains hazy. In the present study, the regulatory mechanism of the miR-216a-mediated inflammatory response in teleost fish was addressed. We found that the expression of miR-216a could be significantly upregulated in the miiuy croaker after challenge with Vibrio anguillarum and lipopolysaccharide. Bioinformatics predictions demonstrated a potential binding site of miR-216a in the 3' untranslated region of the p65 gene, and the result was further confirmed by luciferase assay. Moreover, both the mRNA and protein levels of p65 in macrophages were downregulated by miR-216a. Deletion mutant analysis of the miR-216a promoter showed that the Ap1 and Sp1 transcription factor binding sites are indispensable for the transcription of miR-216a. Further study revealed that overexpression of miR-216a suppresses inflammatory cytokine expression and negatively regulates NF-κB signaling, which inhibit an excessive inflammatory response. The collective results indicate that miR-216a plays a role as a negative regulator involved in modulating the bacterium-induced inflammatory response.

Comparative transcriptome and translatome analysis in contrasting rice genotypes reveals differential mRNA translation in salt-tolerant Pokkali under salt stress.

BACKGROUND: Soil salinity is one of the primary causes of yield decline in rice. Pokkali (Pok) is a highly salt-tolerant landrace, whereas IR29 is a salt-sensitive but widely cultivated genotype. Comparative analysis of these genotypes may offer a better understanding of the salinity tolerance mechanisms in rice. Although most stress-responsive genes are regulated at the transcriptional level, in many cases, changes at the transcriptional level are not always accompanied with the changes in protein abundance, which suggests that the transcriptome needs to be studied in conjunction with the proteome to link the phenotype of stress tolerance or sensitivity. Published reports have largely underscored the importance of transcriptional regulation during salt stress in these genotypes, but the regulation at the translational level has been rarely studied. Using RNA-Seq, we simultaneously analyzed the transcriptome and translatome from control and salt-exposed Pok and IR29 seedlings to unravel molecular insights into gene regulatory mechanisms that differ between these genotypes. RESULTS: Clear differences were evident at both transcriptional and translational levels between the two genotypes even under the control condition. In response to salt stress, 57 differentially expressed genes (DEGs) were commonly upregulated at both transcriptional and translational levels in both genotypes; the overall number of up/downregulated DEGs in IR29 was comparable at both transcriptional and translational levels, whereas in Pok, the number of upregulated DEGs was considerably higher at the translational level (544 DEGs) than at the transcriptional level (219 DEGs); in contrast, the number of downregulated DEGs (58) was significantly less at the translational level than at the transcriptional level (397 DEGs). These results imply that Pok stabilizes mRNAs and also efficiently loads mRNAs onto polysomes for translation during salt stress. CONCLUSION: Under salt stress, Pok is more efficient in maintaining cell wall integrity, detoxifying reactive oxygen species (ROS), translocating molecules and maintaining photosynthesis. The present study confirmed the known salt stress-associated genes and also identified a number of putative new salt-responsive genes. Most importantly, the study revealed that the translational regulation under salinity plays an important role in salt-tolerant Pok, but such regulation was less evident in the salt-sensitive IR29.

The evolution and functional characterization of miiuy croaker interferon regulatory factor 9 involved in immune response.

Interferon regulatory factors (IRFs) are transcription factors which play important roles in regulating the expression of type I interferons (IFNs) and IFN-stimulated genes. IRF9 is one of the IRF family gene members which belongs to the IRF4 subfamily. Mammalian IRF9 has been known to be involved in antiviral responses as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex. In fish, only a few studies investigated the characteristics of IRF9 and the role in IFN signaling. In this study, we identified the IRF9 gene from miiuy croaker (mmiIRF9) and studied its feature and function. Sequence analysis showed the similarity of mmiIRF9 and other fish IRF9 genes. Structural and syntenic analysis showed the conservatism in fish IRF9 genes. The result of expression analysis in normal tissues and infected tissues and macrophages showed that mmiIRF9 expressed in all tested normal tissues and up-regulated expression in liver, kidney and macrophages after stimulated with poly(I:C). Luciferase reporter assays demonstrated the mmiIRF9 can induced IFNα and IFNß luciferase reporters and the cellular localization of mmiIRF9 was mainly distributed in the cytoplasm in Hela cells. Furthermore, the evolutionary analysis of IRF4 subfamily showed the IRF4 and IRF8 may be the most ancient and conservative genes in the evolution of this subfamily.

miRNA-8159 is involved in TLR signaling pathway regulation after pathogen infection by direct targeting TLR13 in miiuy croaker.

Toll-like receptors (TLRs) play a crucial role in the recognition of immune reactions against invading pathogens. The molecular regulation mechanisms of TLR expression in aquatic organisms remain unclear. MicroRNAs (miRNAs) are small non-coding RNAs that are critical adjustors of immune signaling pathway at the post-transcriptional level and play critical roles in intricate networks of host-pathogen interactions and innate immunity. The critical role of TLRs in host defense for discerning certain kinds of pathogen associated molecular patternsand striking a cascade immune response in fish have been demonstrated. Miiuy croaker TLR13 significantly increased after infection with Vibrio anguillarum, which suggests that mmiTLR13 plays an important role in innate immunity. In this study, the role of miR-8159 was explored in regulating TLR13, which is involved in inflammatory responses in miiuy croakers. Bioinformatics was used to predict miR-8159, which has a direct negative regulatory effect on TLR13 in miiuy croaker. Afterward, the dual luciferase reporter assay containing miRNA mimics or inhibitors and pre-miR-8159 showed that miR-8159 was the direct negative regulator of TLR13 in miiuy croaker. Moreover, miR-8159 downregulated the expression of TLR13 in the transcription level. The expression of miR-8159 could be upregulated by V. anguillarum challenged miiuy croaker and LPS exposure macrophages. Thus, miR-8159 could be induced by V. anguillarum and may function as a negative regulator of TLR13 in the immune response of miiuy croakers.

Functional characterization and subcellular localization of miiuy croaker cytosolic MITA involved in activation NF-κB pathway.

In the innate immune responses in host protection, pattern recognition receptors are involve in a variety of sensing mechanisms to recognize and counter pathogen invasion. Recently, a resident endoplasmic reticulum adaptor, stimulator of interferon genes (STING) protein, also called MPYS, ERIS and MITA, has been indicated to play a critical role in innate immune responses. In this study, bioinformatics and functions of MITA from miiuy croaker (mmiMITA) were characterized. MmiMITA was ubiquitously expressed in the detected tissues of miiuy croaker and the highest expression in liver. Moreover, the expressions were dramatically upregulated in liver, spleen and kidney after stimulation with poly(I:C). Meanwhile, the expressions analysis of mmiMITA at the transcriptome database further prove that upon different stimuli, mmiMITA is most sensitive to the stimulation of poly(I:C) in vivo. Furthermore, the immunofluorescence of mmiMITA shows in the cytoplasm of Hela cells. Overexpression of mmiMITA can activate NF-κB reporter gene, it implying that mmiMITA might act as an important role in immune responses by activating NF-κB to induce the production of pro-inflammatory cytokines. The research of mmiMITA will enrich the information of teleost fish MITA and the functional experiments also will be helpful for researching about fish immune systems in the future.

miR-122 involved in the regulation of toll-like receptor signaling pathway after Vibrio anguillarum infection by targeting TLR14 in miiuy croaker.

Innate and acquired immune responses provide critical line in defense against pathogens. MicroRNAs (miRNAs) play crucial roles in regulate gene expression in inflammation and innate immunity. However, the main role of miRNAs are in a large part unclear in aquatic organisms. In this study, we found that miR-122 displayed dramatically declined expression profiles in Vibrio anguillarum challenged miiuy croaker. Meanwhile, we have explored TLR14 as a novel target gene of miR-122 involved in miiuy croakers inflammatory and immune response, which were further estimated through negative expression profiles in both Vibrio anguillarum challenged miiuy croaker and LPS exposure macrophages. Finally, in the dual-luciferase reporter assay presented mmi-miR-122 negatively regulated the 3'-UTR of wild-type in luciferase activity rather than the mutant one in HEK-293T cells. This result demonstrated that mmi-miR-122 modulated TLR14 expression by directly targeting TLR14-3'UTR. All of the present data suggested miR-122 was involved in TLR cascade could modulate Vibrio anguillarum infection in miiuy croakers.

Long Noncoding RNA MIR122HG Inhibits MAVS-Mediated Antiviral Immune Response by Deriving miR-122 in Miiuy Croaker (Miichthys miiuy).

Long noncoding RNAs (lncRNAs) function as micro regulators to impact gene expression after multiple pathogen infections, which have been largely studied in the last few years. Although lncRNA studies on lower vertebrates have received less attention than those on mammals, current studies suggest that lncRNA plays an important role in the immune response of fish to pathogen infections. Here, we studied the effect of MIR122HG as the host gene of miR-122 and indirectly negatively regulate MAVS-mediated antiviral immune responses in miiuy croaker (Miichthysmiiuy). We found that poly(I:C) significantly increases the host MIR122HG expression. The increased MIR122HG expression inhibited the production of the antiviral immune-related genes IFN-1, ISG15 and Viperin upon SCRV treatment. In addition, MIR122HG can act as a pivotally negative regulator involved in the MAVS-mediated NF-κB and IRF3 signaling pathways, which can effectively avoid an excessive immune response. Additionally, we found that MIR122HG can promote the replication of SCRV. Our study provides evidence about the involvement of lncRNAs in the antiviral immune response of fish and broadens the understanding of the function of lncRNAs as a precursor miRNA in teleost fish.

Zw10 negatively regulates the MyD88-mediated NF-κB signaling through autophagy in teleost fish.

MyD88 is a typical street protein of the TLRs signaling pathway and is a central player in innate immune signaling, which can regulate the NF-κB signaling pathway and promote downstream inflammatory factors. However, studies on the molecular mechanisms of the MyD88-mediated NF-κB signaling pathway in teleosts have been poorly reported. In this study, we report that Zw10 targets MyD88 to inhibit NF-κB activation. Zw10 inhibits cell proliferation and MyD88-mediated innate immunity in fish. Zw10 interacts with MyD88, and its Δ2 domain is very critical for MyD88 degradation. In addition, we found that Zw10 degrade MyD88 by autophagy, thereby negatively regulating the MyD88-mediated NF-κB signaling pathway. This study not only enriches the research on the innate immunity of teleost fish, but also provides insights for the regulating mechanism for mammals.

Characterization of MDA5 and microRNA-203 negatively regulates the RLR signaling pathway via targeting MDA5 in miiuy croaker.

MDA5 is a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors), which may play a crucial role in the immune regulation process. Recently, microRNAs (miRNAs) have been shown to act as an important regulator in the RLRs signaling pathway. Additionally, the MDA5 gene, as a significant cytosolic pathogen recognition receptor (PRR), its characteristics and functions have been extensively investigated, while less research has been done on the mechanisms of MDA5-miRNA mediated gene regulation. In this study, the evolution and functional characterization of MDA5 from miiuy croaker (mmiMDA5) were characterized. Comparative genomic analysis demonstrated that the ascidiacea and superclass do not have the MDA5 gene in the process of evolution. MDA5 contains four structural domains: CARD, ResIII, Helicase C, and RIG-I C-RD. The MDA5 was ubiquitously expressed in all tested miiuy croaker tissues. Moreover, the expressions were significantly up-regulated after stimulation with poly (I: C), which indicated that MDA5 might be involved in the antiviral immune response. The bioinformatics predicted programs have indicated that miR-203 has a direct negative regulatory effect on MDA5 in miiuy croaker. Furthermore, the dual-luciferase reporter assay have showed that miR-203 was the direct negative regulator of MDA5 in miiuy croaker. This study is the first to demonstrate that miRNA can suppress cytokines by regulating the RLR signaling pathway in teleost fish, providing some new ideas for studying miRNA-mediated regulation of immune responses in mammals.