Miiuy Croaker Research Articles

Preparation, Physicochemical and Antioxidant Properties of Acid- and Pepsin-Soluble Collagens from the Swim Bladders of Miiuy Croaker (Miichthys miiuy).

Collagen is one of the most useful biomaterials and widely applied in functional food and cosmetics. However, some consumers have paid close attention to the safety of mammalian collagens because of the outbreaks of bovine spongiform encephalopathy (BSE), foot-and-mouth disease (FMD), and other prion diseases. Therefore, there is a strong demand for developing alternative sources of collagen, with one promising source being from the process by-products of commercial fisheries. In this report, acid-soluble collagen (ASC-SB) and pepsin-soluble collagen (PSC-SB) from swim bladders of miiuy croaker (Miichthys miiuy) were isolated with yields of 1.33 ± 0.11% and 8.37 ± 0.24% of dry swim bladder weight. Glycine was the major amino acid present, with a content of 320.5 (ASC-SB) and 333.6 residues/1000 residues (PSC-SB). ASC-SB and PSC-SB had much lower denaturation temperatures compared to mammalian collagen, a consequence of low imino acid contents (196.7 and 199.5 residues/1000 residues for ASC-SB and PSC-SB, respectively). The data of amino acid composition, SDS-PAGE pattern, UV and FTIR spectra confirmed that ASC-SB and PSC-SB were mainly composed of type I collagen. FTIR spectra data indicated there were more hydrogen bonding and intermolecular crosslinks in ASC-SB. These collagens showed high solubility in the acidic pH ranges and low NaCl concentrations (less than 2%). The Zeta potential values of ASC-SB and PSC-SB were 6.74 and 6.85, respectively. ASC-SB and PSC-SB presented irregular, dense, sheet-like films linked by random-coiled filaments under scanning electron microscopy. In addition, ASC-SB and PSC-SB could scavenge DPPH radical, hydroxyl radical, superoxide anion radical, and ABTS radical in a dose-dependent manner. Overall, the results indicate that collagens from the swim bladders of miiuy croaker are a viable substitute for mammalian collagen, with potential functional food and cosmeceutical applications.

MicroRNA-375 modulates the NF-κB pathway in miiuy croaker by targeting DUSP1 gene

microRNAs (miRNAs) are highly conserved, small non-coding endogenous molecule, and can participate in a variety of biological processes in organisms such as development, growth and immune response. Dual-Specificity Phosphatases (DUSPs) are enzymes that can remove phosphate groups from phosphatases. Research found that DUSP1 is an important molecule in the process of MAPK regulation. However, as a significant regulatory factor, the study of DUSP1 was very few in fish. Consequently, in this study, the regulatory role of miRNAs on DUSP1 has been verified through dual-luciferase reporter assay and western blotting analysis. Furethermore, we found that miR-375 mimics and pre-miR-375 plasmid can negatively regulate the target gene DUSP1 in miiuy croaker through combining with 3'untranslated region of DUSP1 gene. These experiment results directly indicate the negative regulatory function of miR-375 to DUSP1. Moreover, miR-375 can negatively regulate NF-κB signaling pathway via target to DUSP1. This study can increase our knowledge and help us to understand complexity of genomic and complex gene expression regulatory networks in teleost fish.

MicroRNA-210 participates in regulating RIG-I signaling pathway via targeting DUBA in miiuy croaker after poly(I:C) stimulation

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that participate in the regulation of various biological processes. A series of microRNAs have been shown to be important regulators of both innate and adaptive immune responses, including RIG-I signaling pathway. In this study, we evaluated the regulation role of miR-210 in the RLRs signaling pathway of miiuy croaker. Upon poly(I:C) stimulation, the expression of miR-210 in both miiuy croaker spleen tissues and macrophages were significantly upregulated. By means of the dual luciferase reporter assay, a direct interaction between miR-210 and the 3-untranslated region (UTR) of Deubiquitinating enzyme A (DUBA) was confirmed, and we found that miR-210 could reduce the luciferase levels of wild-type DUBA 3′UTR, whereas mutant-type led to a complete abrogation of the negative effect. Furthermore, the negative regulatory effects of pre-miR-210 on DUBA have been indicated in a dose- and time-dependent manners. As DUBA is an important regulator involved in the RLRs signaling pathway and could bind with and regulate TRAF3, we also examined the expression patterns of DUBA and TRAF3 in vivo and in vitro. We found that the expression of both DUBA and TRAF3 were significantly changed upon poly(I:C) stimulation in miiuy croaker. The expression patterns between miR-210 and DUBA showed a negative correlation, which indicated that miR-210 can target and downregulate the expression of DUBA. Overall, these results will enrich the knowledge of immune response related miRNAs in miiuy croaker, which will be useful for better understanding the complicated regulatory networks in fish species.

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.

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.

Identification and functional characterization of interferon regulatory factor 7 involved in activation JAK/STAT pathway in miiuy croaker

Interferon regulatory factor (IRF) family is a transcription factor family which plays an important role in the regulation of natural immunity and immune cell differentiation. IRF7 is important to regulate the response of type I interferon (IFN) to viral infection. Thus, more researches of the characteristic and functions of IRF7 should be done to get better understanding of the mechanisms underlying immune reactions. Here, the characterization of full-length cDNA of IRF7 was reported from miiuy croaker. Gene characterization analysis of mmiIRF7 showed conservative with other fish and inferred that the difference of tryptophan residues in IRF7 may occurred in the period of fish-specific genome duplication (FSGD) or earlier. Syntenic analysis of IRF7 showed that fish IRF7 had more highly conserved synteny than the higher vertebrates IRF7. Luciferase reporter assays result showed the ability of mmiIRF7 for activation of IFNα, IFNβ, IFNγ and ISRE luciferase reporter. In this study, we systematically and comprehensively analyzed evolution and function of mmiIRF7, which will provide the basis for future research on fish IRF family.

Characterization and role of suppressor of cytokine signaling 1a (SOCS1a) in a teleost fish, Miichthys miiuy

The suppressor of cytokine signaling 1 (SOCS1) is a crucial regulator in the immune systems of mammals, which functions classically as a negatively regulator in the IFN signaling pathways. However, data on functional characterization of SOCS1 in lower vertebrates is limited. In this study, we identified and characterized the full-length SOCS1a gene of miiuy croaker (Miichthys miiuy). The sequence analysis results showed that miiuy croaker SOCS1a (mmSOCS1a) shared some conserved motifs with other vertebrates. To further study the function of fish SOCS1, we identified mmSOCS1a and determined its potential ability to perceive poly(I:C) stimulation. Induction experiments with poly(I:C) indicated the significant expression levels of mmSOCS1a in liver and kidney. In addition, mmSOCS1a could inhibit poly(I:C)-induced or IFNs-induced ISRE reporter gene activation. In a word, we systematically and comprehensively analyzed evolution and function of mmSOCS1a, which will provide the basis for future research on fish SOCS family.

Inducible microRNA-122 modulates RIG-I signaling pathway via targeting DAK in miiuy croaker after poly(I:C) stimulation

MicroRNA-122 (miR-122) was originally identified in mouse and then lots of researches on miR-122 had been performed in mammals. However, the functional study of miR-122 were restricted in fish. In miiuy croaker, miR-122 is sensitive to poly(I:C) stimulation. In this study, a combination of bioinformatics and experimental techniques were used to investigate the functions of miR-122. DAK is a putative target gene of miR-122 which was predicted by bioinformatics, and further the luciferase reporter assays were used to confirm the target sites in DAK 3'untranslated region. The inhibiting effect of miR-122 mimics or pre-miR-122 on DAK presented the dose and time dependent manners, and the pre-miR-122 showed stronger inhibiting effect on DAK than the miR-122 mimics. Therefore, the miR-122 participate in regulating RIG-I-like receptors signaling pathway via inhibiting DAK which is the inhibitors of MDA5. The expression of miR-122 and DAK showed negative relationship in both miiuy croaker spleen and macrophages, which imply that miR-122 may regulate DAK at the post-transcriptional level. These results will enhance our understanding about the regulation of miRNAs on immune response in fish.

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.

Cloning, expression pattern and promoter functional analysis of cyp19a1a gene in miiuy croaker

Gonadal-specific aromatase encoded by cyp19a1a is the important enzyme controlling estrogen biosynthesis in teleosts. In the present study, the cDNA sequence of cyp19a1a was cloned and characterized from miiuy croaker Miichthys miiuy. The cDNA encoded a protein of 519 amino acids with five structural regions. Higher identities of amino acid sequences and conserved structural regions were found between Mmcyp19a1a and other cyp19a1a genes. In addition, Mmcyp19a1a was clustered together with other seawater fishes. Immunohistochemical analysis revealed that Mmcyp19a1a was localized exclusively in the cytoplasmic of thecal and granulosa cells surrounding the oocytes. Both the protein and mRNA levels of Mmcyp19a1a were increased significantly at the stage III follicles (mid-vitellogenic) and then decreased along with vitellogenesis.Interestingly, strong immunoreactive signals were also detected in the supporting cells of connective tissues during ovarian development. A 1777bp promoter fragment of Mmcyp19a1a was also isolated, and functional analysis using an EGFP reporter fusion in zebrafish larvae presented positive signals in the above of yolk sac, where is the region of pronephros and germ plasm occur. The Mmcyp19a1a:EGFP expression pattern was generally consistent with the endogenous cyp19a1a genesis. These results indicate that the Mmcyp19a1a gene plays an important role during vitellogenesis and oocyte maturation. The constructor of Mmcyp19a1a:EGFP may provide a useful tool for genetic analysis of gonad development in teleost.

NOD1 is the innate immune receptor for iE-DAP and can activate NF-κB pathway in teleost fish

The innate immune system is the first line for organisms defense against microbial infection, and NOD-like receptors (NLRs) protein family is an important member of innate immunity effector molecules. It has been proved that NLRs are located in the endochylema and can senses of microbial products. NOD1 is one of the representatives of this family, it has been proved that in mammals, NOD1 can distinguish a specific muropeptide (G-d-glutamyl-meso-diaminopimelic acid, iE-DAP) which was derived from bacterial peptidoglycans. However, the NOD-mediated intracellular recognition of microorganisms remains largely uncharacterized in teleost fishes. In this study, we use miiuy croaker (Miichthys miiuy) as a model to determine NOD1 can response to the infection of Gram-negative bacteria and it is the receptor that can recognize of iE-DAP by LRRs domain, it can activate the NF-κB signaling pathway through recruit RIP2 to induce inflammatory response in teleost fishes. Results showed that NOD1 can recognize the components of Gram-negative bacteria and activate inflammatory response to resistance of bacterial infection. Our study can improve the knowledge on immune system of fishes and provide a theoretical basis for the study of prevention and treatment of fish diseases.

MicroRNA-148 as a negative regulator of the common TLR adaptor mediates inflammatory response in teleost fish

MicroRNAs are small endogenous noncoding RNAs implicating in the regulation of diverse biological processes, including proliferation, differentiation, cancer, apoptosis, and viral infections. MicroRNAs regulate gene expression by either mRNA degradation or inhibition of protein translation. Although microRNAs have emerged as important controller involved in regulation of inflammatory response, the microRNA-mediated regulatory mechanism remains less clear in teleost. Here, we report that miR-148 targets MyD88 and down-regulates its expression by inhibition protein translation rather than degradation mRNA in miiuy croaker. Additionally, we found that miR-148 was significantly upregulated in miiuy croaker after treated with Vibro harveyi, as well as LPS. Overexpression of miR-148 inhibited LPS-induced inflammatory cytokines production, such as IL-6 and IL-1β, which then avoid excessive inflammation response. miR-148 has also been identified to suppress NF-κB pathway through targeting and repressing MyD88 expression. Taken together, our findings indicate that miR-148 participates in bacteria-induced inflammatory response and act as a negative regulator for MyD88-mediated NF-κB signaling, which may clarify the mechanism of microRNAs for avoiding excessive inflammation in teleost fish.

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.

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 IFNβ 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.

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.

Genome-guided transcriptome analysis of miiuy croaker provides insights into pattern recognition receptors and cytokines in response to Vibrio anguillarum

Miiuy croaker (Miichthys miiuy), as an economically important marine fish is affected by numerous bacterial diseases. Infection by bacterial pathogen Vibrio anguillarum causes high mortality and great economic loss in aquaculture. To understand the immune response of the miiuy croaker to V. anguillarum infection, we analyzed the transcriptomic profile of V. anguillarum-challenged M. miiuy, and two cDNA libraries, namely, the normal library (Ctrl) and V. anguillarum challenged library (Van) were constructed. Combined with the whole genome-guided assembly, total 47,971 unigenes were acquired. Moreover, 2482 significantly differentially expressed unigenes were identified based on the expression patterns. Immune-related differentially expressed genes (DEGs) that were significantly up- or down-regulated after V. anguillarum injection were identified via enrichment analysis using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. To further understand the expression profiles that underlie the response to V. anguillarum infection, we investigated the DEGs that are related to pattern-recognition receptors and cytokines. The expression patterns of relevant DEGs were validated by qRT-PCR. Additionally, the expression profiles of DEGs that are related to pattern-recognition receptors and cytokines were further measured in miiuy croaker macrophages. This study is the first to identify and characterize the transcriptome of miiuy croaker in response to V. anguillarum infection. The results expand the general understanding of the immune defense mechanisms of miiuy croaker and provide helpful information for counteracting V. anguillarum infection.

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.

Characterization of IL-1β and two types of IL-1 receptors in miiuy croaker and evolution analysis of IL-1 family

Interleukin (IL)-1β is a prototypical proinflammatory cytokine that belongs to the IL-1 family. This cytokine possesses two receptor types, namely, IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII). IL-1RI, is an IL-1 receptor that plays a crucial role in immune responses and IL-1RII is a decoy receptor for IL-1β signaling inhibitors in mammals. IL-1β, together with its two types of receptors, has been characterized in mammals and implicated in immunity. However, IL-1β and IL-1 receptors in teleost species have been rarely investigated. In this study three genes, namely, IL-1β, IL-1RI, and IL-1RII, were identified and characterized from miiuy croaker. Structural and comparative analysis revealed that miiuy croaker IL-1β, IL-1RI and IL-1RII, particularly their functional domains, were conservative in most of the species. Simultaneously, synteny phylogenetic analysis indicated that IL-1β and IL-18 were widely distributed in vertebrates and hence might be the ancestors of the IL-1 family. Challenge experiment demonstrated that IL-1β, IL-1RI and IL-1RII expression in miiuy croaker was induced by LPS and poly (I:C). IL-1RI expression was also induced by the overexpressed miiuy croaker IL-1β protein which in cell supernatant, whereas IL-1RII was not induced.

MicroRNA-145 regulates the RLR signaling pathway in miiuy croaker after poly(I:C) stimulation via targeting MDA5

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that participate in diverse biological processes via degrading the target mRNAs or repressing translation. In this study, the regulation of miRNA to the RLR (RIG-I-like receptor) signaling pathway by degrading the target mRNAs was researched in miiuy croaker. MDA5, a microRNA-145-5p (miR-145-5p) putative target gene, was predicted by bioinformatics, and the target sites from the 3'untranslated region of MDA5 transcripts were confirmed using luciferase reporter assays. Pre-miR-145 was more effective in inhibiting MDA5 than miR-145-5p mimic, and the effect was dose- and time-dependent. The expression patterns of miR-145-5p and MDA5 were analyzed in liver and kidney from miiuy croaker. Results implied that miR-145-5p may function via degrading the MDA5 mRNAs, thereby regulating the RLR signaling pathway. Studies on miR-145-5p will enrich knowledge of its functions in immune response regulation in fish, as well as offer a basis for regulatory networks that are composed of numerous miRNAs.

Up-regulated of miR-8159-5p and miR-217-5p by LPS stimulation negatively co-regulate TLR1 in miiuy croaker

Toll-like receptors (TLRs) are a group of pattern-recognition receptors which play vital roles in ligand recognition and activation of the innate immune response. As an important member of TLRs family, TLR1 is mainly responsible for PAMPs from bacteria and play a pivotal role in sensing microbial products. Recent studies revealed that TLR1 could perceive LPS stimulation and transfer signals to activate the NF-κB pathway, whereas ligands and signaling pathway of TLR1 are still unclear in fish. Growing evidence has shown that miRNAs (microRNAs) play as negative regulators in controlling the diverse of biophysical and biochemical processes at the post-transcriptional level. In this study, we used a combination of bioinformatics and experimental techniques to exhibit that both miR-8159-5p and miR-217-5p were the direct negative regulators of TLR1 in miiuy croaker. Furthermore, dual-luciferase reporter assays showed that combining miR-8159-5p and miR-217-5p exhibited a greater negative regulatory effect on TLR1 than only miR-8159-5p or miR-217-5p. Additionally, we also demonstrated that the expression of both the two miRNAs could be up-regulated by LPS stimulation in either LPS-stimulation spleen tissue or LPS-treated cultured macrophage, which indicating that miR-8159-5p and miR-217-5p could be induced by LPS and may be as the negative regulators of TLR1 involved in the immune response to LPS stimulation. These results would enhance our understanding of the miRNA regulation in fish TLR signaling pathways.