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.

Innate immunity is evolutionarily conserved and acts as the first line of host defense. Recognition of microbial pathogens is an essential process for the initiation of innate immune responses and is mediated by numerous germline-encoded pattern-recognition receptors (PRRs) 2 (1,2). TLR-like receptors (TLRs), one of the best-characterized PRRs, initiate a wide spectrum of responses from phagocytosis to production of cytokines, which further shape and enhance the inflammatory and adaptive immune responses (3). TLRs trigger the activation of intracellular signaling through a cytoplasmic myeloid differentiation primary response protein 88 (MyD88)-dependent pathway or a MyD88-independent pathway. All mammalian TLRs, with the exception of TLR3, depend at least in part on MyD88 adaptor to transmit signals. In the MyD88-dependent signaling pathway, MyD88 recruits the interleukin-1 receptor-associated kinases (IRAKs) complex, which includes four subunits: two active kinases (IRAK1 and IRAK4) and two noncatalytic subunits (IRAK2 and IRAKM) (4). Once IRAK1 is phosphorylated, IRAKs associate with TNF receptor-associated factor 6 (TRAF6), leading to activation of NF-B and MAPKs signaling (5)(6)(7). The activation of NF-B and MAPKs then induces the transcription of various inflammatory genes, including IL-1␤, IL-6, and TNF␣, leading to inflammatory and innate immune responses (7).
Given that TLRs play vital roles in detecting pathogens and initiating inflammatory responses, strict regulation of the TLR signaling pathways is, therefore, important. Any dysregulation of TLR signaling will disrupt immune homeostasis, which may further induce autoimmune and inflammatory diseases (8). Many molecules have been demonstrated as positive or negative regulators of TLR signaling, including phosphatases, protein kinases, ubiquitin-related proteins, membrane molecules, endosome/lysosome-localized molecules, gene transcription coactivators, antigen-presenting molecules, and even HSP70, HSP70L1, and NGF (9). Nevertheless, the full anti-inflammatory response mechanisms and the processes by which inflammation are resolved remain incompletely understood.
Recently, microRNAs (miRNAs) have emerged as important controllers involved in innate immune response. miRNAs are a class of endogenous, single-stranded, conserved, noncoding small RNAs consisting of ϳ22 nucleotides. The small RNAs are encoded in genomic clusters and produced by an elaborate expression and processing mechanism (10,11). The mature miRNA uses its "seed sequence" to bind partially complementary sequences in the 3Ј-untranslated region (UTR) of target mRNA transcripts, leading to mRNA degradation or translational repression (11). Through regulating gene expression, miRNAs are implicated in regulating diverse biological processes, including embryogenesis, differentiation, tumorigenesis, inflammation, and immunity (12). Presently, more than 1,000 miRNAs have been identified in the human genome, and as much as 60% of all mRNAs have been predicted to be regulated by miRNAs to some extent (13). It is not surprising that miRNAs have been implicated in regulating the components of TLR signaling pathways. For instance, miR-233 and miR-146a have been shown to negatively regulate TLR expression (14), and miR-155 has also been reported to target MyD88 and TBK2 (15,16).
The Gram-negative bacterium, Vibrio harveyi, is a typical Gram-negative pathogen for a wide range of marine animals, and can lead to a variety of vibriosis, a common aquatic animal disease associated with high mortality throughout the world (17). Recently, the development of the miiuy croaker (Miichthys miiuy) aquaculture has been hindered by V. harveyi, leading to high mortality. Bacterial infection can cause a severe inflammatory response and significant pathology. The role of miRNAs in bacteria-triggered inflammatory response has not been investigated in detail, especially in fish species. Here, we found that the expression of miR-214 is significantly up-regulated following stimulation with V. harveyi and LPS in miiuy croaker. Overexpression of miR-214 down-regulates TNF-␣, IL-6, IL-1␤, and IL-10 expression levels in LPS-exposed macrophages. Further investigation revealed that miR-214 suppresses myd88 expression to regulate the NF-B signaling pathway and inflammatory cytokines, thereby avoiding excessive inflammation. These studies indicate that miR-214 is up-regulated upon V. harveyi infection and acts as a negative regulator of NF-B signaling by targeting myd88.

V. harveyi and LPS stimulation induce miR-214 expression
To examine miRNAs that are potentially involved in the regulation of V. harveyi infection, a small RNA deep-sequencing analysis of miiuy croaker spleen challenged with V. harveyi was performed. From the deep-sequencing data, we selected several miRNAs that were differentially expressed upon stimulation. Among those miRNAs, miR-214 was significantly up-regulated following V. harveyi stimulation (data not shown). To further investigate expression profiles of miR-214 upon stimulation, the expression of miR-214 in V. harveyi-infected miiuy croaker liver was determined at different times post-infection (Fig. 1A).
LPS, the endotoxin of Gram-negative bacteria, contributes greatly to the structural integrity of the bacteria and elicits strong immune responses in animals. We thus wanted to explore whether miR-214 expression was regulated by LPS stimulation, we used LPS to stimulate miiuy croaker. As shown in Fig. 1B, miR-214 expression was up-regulated quickly from 12 to 48 h and peaked at 36 h after LPS stimulation. Taken together, the up-regulation of miR-214 upon stimulation of V. harveyi and LPS suggested that miR-214 may function as a regulator involved in the regulation of the immune response.

miR-214 suppresses inflammatory cytokines production
To further determine whether miR-214 expression upon bacteria stimulation could affect the immune response in fish, we have explored the role of miR-214 in inflammatory cytokine production through transfection with miR-214 mimics and miR-214 inhibitor into miiuy croaker macrophages. miRNAs mimics could stimulate naturally occurring mature miRNAs, whereas miRNAs inhibitors blocks the activity of endogenous miRNAs by complementarity. As shown in Fig. 2A, transfection of miR-214 mimics increased miR-214 expression ϳ800-fold in macrophages, whereas miR-214 inhibitor decreased its expression over 55%. Then, we assessed the contribution of miR-214 mimics and miR-214 inhibitor to the regulation of inflammatory cytokine gene expressions after LPS stimulation. To this end, macrophages were transfected with miR-214, control miRNAs, miR-214 inhibitor, and control inhibitor, respectively, for at least 24 h before LPS simulation. As shown in Fig.  2B, compared with control miRNA, overexpression of miR-214 significantly reduced the mRNA levels of TNF-␣, IL-6, IL-1␤, and IL-10 in LPS-exposed macrophages. By contrast, the miR-214 inhibitor caused the enhancement of TNF-␣, IL-6, IL-1␤, and IL-10 expression (Fig. 2C). These results demonstrated that miR-214 suppresses the production of inflammatory cytokines, including TNF-␣, IL-6, IL-1␤, and IL-10 in macrophages after LPS stimulation, suggesting miR-214 may play a negative role in response to LPS challenge.

miR-214 regulates the components of the myd88-dependent signaling cascade
Upon recognition of infectious pathogens, MyD88 is recruited to all TLRs (except for TLR3) and associates with IRAKs and TRAF6 to transducer signals (20,21). In mammals, TLR4 recognizes LPS and mediates the MyD88-dependent pathway (22). Although TLR4 in fish is lost or fails to recognize LPS (23), other TLRs may substitute for this function because TLRs in fish display a high variety and distinct features. Compelling evidence shows that signal transduction of the TLR pathway is highly conserved from invertebrates to mammals (24,25). Therefore, we evaluated proteins in the myd88-dependent signaling cascade to identify the potential target of miR-214. We thus transfected miR-214, control miRNAs, miR-214 inhibitor, and control inhibitor, respectively, into macrophages for up to 24 h, and then monitored mRNA levels of myd88, IRAK1, IRAK4, and TRAF6 by quantitative RT-PCR. As shown in Fig. 3, the expressions of myd88, IRAK1, IRAK4, and TRAF6 were decreased by miR-214 overexpression, whereas increased by miR-214 inhibitor treatment in comparison with the control group. Thus, these results demonstrated that miR-214 suppresses the expression of genes involved in the myd88-dependent pathway cascade.

myd88 is a target of miR-214
To further characterize the potential target of miR-214, we used miRNA target prediction program TargetScan (26,27) to search for potential miR-214 targets, identifying a putative miR-214 binding site in the 3ЈUTR of myd88 (Fig. 4A). To certify the prediction, we constructed reporter plasmids by cloning miiuy croaker myd88-3ЈUTR into the pmirGLO luciferase reporter vector within the mutation at the miR-214 binding site as control, and then transfected the plasmids into HEK293 cells for Dual-Luciferase reporter assays. As shown in Fig. 4B, the relative luciferase activity was reduced by 62% following cotransfection with miR-214 mimics compared with transfection with control miRNAs (miR-Ctrl). By contrast, no change of luciferase was observed in cells trans-fected with mutant-type constructs. This result was subsequently confirmed by concentration and time gradient experiments (Fig. 4D). Moreover, as shown in Fig. 3C, miR-214 mimics and inhibitor were used to further verify the down-regulation mechanism, and results revealed that inhibition of luciferase activity was attenuated after cotransfection with the miR-214 inhibitor.  In addition, pre-miR-214 was predicted, and its sequence was cloned into the pcDNA6.2-GW/EmGFP vector to construct the pre-miR-214 plasmid (Fig. 4A). Subsequently, we sought to transfect the pre-miR-214 plasmid, together with wild-type or mutant-type myd88-3ЈUTR into HEK293 cells to demonstrate the down-regulation mechanism. After 48 h, the Dual-Luciferase assays were conducted to measure the relative luciferase activity, which was reduced by 43.6% compared with the control (Fig. 4B). This result was confirmed by concentration and time gradient experiments (Fig. 4E). Furthermore, both miR-214 mimics and pre-miR-214 plasmid down-regulated GFP gene expression when the myd88-3ЈUTR was cloned into the pIZ/EGFP vector in HEK293 cells, whereas no change of GFP gene expression was observed within the mutant-type constructs (Fig. 4F). The above data suggested that miR-214 targets the 3ЈUTR of myd88, and myd88 is a new target of miR-214.

miR-214 decreases the abundance of myd88 at both the mRNA and protein levels
Given that miRNAs down-regulate the target genes through mRNA degradation or inhibition of translation, we next inves-tigated which mechanism results in the suppression of myd88 by miR-214 in fish. To this end, we transfected using miR-214 mimics or control miRNA into miiuy croaker macrophages, and then measured the mRNA and protein levels of myd88. As shown in Fig. 5A, transfection with miR-214 mimics markedly decreased MyD88 abundance at both the mRNA and protein levels in a dose-dependent manner. As a comparison, transfection with the miR-214 inhibitor enhanced myd88 abundance at both the mRNA and protein levels as compared with that in cells transfected with the control inhibitor (Fig. 5B). These results indicated that miR-214 can decrease the abundance of endogenous myd88. Additionally, the myd88 expression plasmid was constructed within the full-length CDS region and 3ЈUTR of the miiuy croaker myd88 gene. Given that the miRNA processing system is conserved from invertebrates to vertebrates (28,29), we then sought to transfect with the myd88 expression plasmid, together with the pre-miR-214 plasmid into HEK293 cells. To construct the myd88 expression plasmid, the full-length CDS region and 3ЈUTR of the miiuy croaker myd88 gene was amplified by specific primer pairs and cloned into pcDNA3.1 vector with FLAG tag. At 48 h post-transfection, the mRNA and protein levels of myd88 were measured, and results indicated that the mRNA and protein abundance of myd88 expression could also be decreased by pre-miR-214 in a dose-dependent manner (Fig. 5C). Taken together, these results demonstrated that miR-214 could decrease myd88 abundance at both the mRNA and protein levels, which may indicate that miR-214 could regulate myd88 expression by translation inhibition and mRNA degradation.

miR-214 suppresses LPS-induced inflammation through myd88
To explore the role of myd88 involved in LPS-induced inflammatory response, we silenced myd88 and then examined the production of inflammatory cytokines in macrophages treated with LPS stimulation. siRNA effectively inhibited the expression levels of myd88 protein and mRNA (Fig. 6, A and B). As shown in Fig. 6C, knockdown of myd88 significantly decreased the levels of TNF-␣ mRNA in macrophages upon LPS stimulation, which produced an effect similar to that of miR-214 overexpression. Similar results were observed among the expression levels of IL-6, IL-1␤, and IL-10 ( Fig. 6, D-F). These results indicated that miR-214 regulates inflammatory response through suppression of endogenous myd88, thereby inhibiting inflammatory cytokine production.

miR-214 suppresses myd88-mediated NF-B pathway
Previous studies demonstrated that compared with other TLR adaptors, the structure of MyD88 is well conserved and its homologs in fish species may function similarly to mammalian counterparts (9,10). In zebrafish, myd88 has been reported to be involved in the positive regulation of the NF-B promoter, and similar reports were also demonstrated in salmonids (11,12). We next investigate the regulatory role of myd88 in miiuy croaker. To this end, the myd88 expression plasmid containing the full-length CDS region and 3ЈUTR of the miiuy croaker myd88 gene was constructed and transfected into HEK293 cells to measure its regulatory function. As shown in Fig. 7A, overexpression of myd88 efficiently up-regulated the NF-B reporter gene. Given that miR-214 targets myd88 and downregulates its expression, we then tested whether miR-214 is a negative regulator in the NF-B pathway. We thus transfected using the myd88 expression plasmid, together with miR-214 mimics and miR-214 inhibitor, into HEK293 cells and each assay was transfected with an equal amount of oligonucleotides. As expected, we found that overexpressed miR-214 could significantly down-regulate the NF-B reporter gene, whereas the down-regulation mechanism was attenuated after cotransfection with miR-214 inhibitor (Fig. 7B). To confirm the above results, the concentration and time gradient experiments were conducted (Fig. 7, C and D). These results indicated that miR-214 suppresses the NF-B pathway by down-regulating myd88. Additionally, a signaling of miR-214 in modulating myd88 expression to down-regulate the NF-B pathway has also been performed (Fig. 7E).

Discussion
miRNAs are involved in the regulation of multiple biological processes, whereas the role of miRNAs in the inflammatory response of fish to bacterial challenge has not been investigated. In this study, we addressed the role of miRNA-regulated pathways in a bacteria triggered inflammatory response. We found that miR-214 could be up-regulated in response to V. harveyi infection, as well as LPS stimulation in fish. Up-regulation of miR-214 suppressed the NF-B pathway by targeting myd88, therefore inhibiting inflammatory cytokines production, including IL-1␤, IL-6, TNF-␣, and IL-10. These findings not only indicate that miR-214 is a new negative regulator of MyD88-meidated immune response but also suggest a novel mechanism for avoiding excessive inflammation in teleost fish.
TLRs are the best-characterized PRRs that detect conserved microbial components during infection and then initiate inflammatory responses. After recognition of the pathogen-associated molecular pattern, TLRs initiate innate immune responses by the MyD88-dependent signaling pathway and MyD88-independent signaling pathway, which then induce Figure 5. miR-214 treatment decreases the abundance of myd88 at both mRNA and protein level. A, the macrophages were transfected with miR-214 or miR-Ctrl within the concentration gradient. After 48 h, myd88 mRNA levels were determined by qPCR and normalized to ␤-actin (left), and myd88 protein levels were determined by Western blot and normalized to ␤-actin (right). B, the macrophages were transfected with miR-214 inhibitor or Ctrlinhibitor within concentration gradient. After 48 h, myd88 mRNA levels were determined by qPCR and normalized to ␤-actin (left), and myd88 protein levels were determined by Western blot and normalized to ␤-actin (right). C, HEK293 cells were cotransfected with myd88 expression plasmid, together with pre-miR-214 and control plasmid. After 48 h, myd88 mRNA levels were determined by qPCR and normalized to ␤-actin (left), and myd88 protein levels were determined by Western blot and normalized to ␤-actin (right). Data are presented as the mean Ϯ S.E. from three independent triplicate experiments. **, p Ͻ 0.01; *, p Ͻ 0.05 versus the controls.
pro-inflammatory cytokine and type I interferon (IFN) production to control inflammatory and immune responses (3,4). However, excessive activation of TLR signaling could disrupt immune homeostasis, thereby inducing some diseases, such as autoimmune diseases, chronic inflammatory diseases, or cancer (8). Therefore, it is especially important to use precise reg-ulation of the TLR signaling pathways. Negative regulators of TLR signaling could intersect at almost every step of the TLR signaling pathway. For instance, NLRX1 was shown to function as intracellular PRRs to suppress TLR signaling through interaction with TRAF6 and IKK complexes (31). With the exception of TLR3, all mammalian TLRs utilize MyD88 to commence  A, HEK293 cells were cotransfectd with myd88 expression plasmid, pRL-TK Renilla luciferase plasmid, together with NF-B reporter genes. After 48 h, the luciferase activity was measured and results presented relative to the luciferase activity in control cell. B, miR-214 or miR-Ctrl and miR-214 inhibitor or Ctrl-inhibitor were cotransfected with myd88 expression plasmid, pRL-TK Renilla luciferase plasmid, together with luciferase reporter NF-B into HEK293 cells. After 24 h, luciferase activity was measured and results presented relative to the luciferase activity in the control cell. C, the concentration gradient experiment of miR-214 (left) or pre-miR-214 plasmid (right) was conducted. After 24 h, the luciferase activity was measured. D, the luciferase activity was measured after 24 or 48 h cotransfection with miR-214 (left) or pre-miR-214 plasmid (right). E, proposed model for miR-214 of inflammatory cytokine secretion via myd88. Data are presented as the mean Ϯ S.E. from three independent triplicate experiments. **, p Ͻ 0.01; *, p Ͻ 0.05 versus the controls. MARCH 31, 2017 • VOLUME 292 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 5287 signaling, activating NF-B and MAPK to regulate host-cell response to pathogens (4). Thus, it may be the most effective to directly regulate MyD88, the TLR downstream adaptor, in host immune response. Accumulated evidence has suggested that MyD88 was strictly regulated during inflammatory response. For instance, IRF4 interacts with MyD88 as a negative regulator of TLR signaling (32).

MicroRNA-214 suppresses NF-B signaling
Recently, miRNAs have emerged as important controllers of innate immune response (33). The present studies showed that miRNAs play vital roles in the regulation of genes of the immune system, including macrophages, microglia, dendritic cells, and T cells (34), and sets of miRNAs have been demonstrated to participate in the modulation of TLR signaling. miRNAs regulate TLR-signaling pathways at multiple layers, including direct regulation of TLR expression, TLR-associated signaling proteins, and TLR-induced transcription factors and functional cytokines. With regard to direct regulation of TLR expression, several miRNAs have been reported. The let-7 miRNA family, including let-7e and let-7i (35), can regulate TLR4 expression, miR-223 is a regulator for both TLR4 and TLR3 (36), and miR-143 can inhibit the expression of TLR2 (37). For the regulation of TLR-associated signaling proteins, recent reports demonstrated that miR-155 can negatively regulate TLR signaling pathways by targeting key signaling proteins, such as MyD88 and TBK2 (15,16). Moreover, miR-146 negatively regulates the MyD88-mediated NF-B pathway post-bacterial infection through targeting IRAR1 and TRAF6 in THP-1 macrophage cells (38). As the important TLR downstream adaptor, MyD88 have also been reported to be regulated by an array of miRNAs, such as miR-146b, miR-155, miR-200b, miR-200c, miR-21, miR-149, and miR-203 (14). Additionally, large amounts of miRNAs are involved in the regulation of transcription factors. For instance, miR-223 targets IKK␣ and miR-199 is a regulator of IKK␤, which all participate in the regulation of NF-B activity (39,40). In accordance with these earlier miRNAs, our findings showed that miR-214 could inhibit the expression of myd88, followed by inhibitory inflammatory cytokine production in miiuy croaker macrophages.
Miiuy croaker, a member of the Sciaenidae family, is an economically important marine fish. Evidence indicates that miiuy croaker has been studied in-depth from transcriptome (41,42), whole-genome (43), to functional genes, which left miiuy croaker as a new model for studying the immune system in fish. V. harveyi, as the marine Gram-negative bacteria, is a serious pathogen for a wide range of marine animals. In response to bacterial infection, innate immunity, and a series of inflammatory response is the main defense system in fish (30). However, dysregulation of inflammatory response may disrupt immune homeostasis, leading to some diseases and even death. In this study, we found that V. harveyi infection could significantly upregulate the expression of miR-214, which could prevent an excessive inflammatory response through inhibiting the production of inflammatory cytokines. These findings demonstrate miR-214 acts as the negative regulator involved in the bacteria-triggered inflammatory response, which can enrich miRNA-mediated feedback on the regulatory mechanisms of the innate signaling pathway.

Sample and challenge
Healthy miiuy croakers (ϳ750 g) were obtained from Zhoushan Fisheries Research Institute (Zhejiang, China) and raised in aerated seawater tanks at 25°C for at least 1 week. For the stimulation experiment, briefly, these healthy fish were randomly divided into two groups in which the experimental individuals were challenged with 1 ml of V. harveyi (1.5 ϫ 10 8 cfu/ml) or 1 ml of suspension of LPS (1 mg/ml, Sigma) intraperitoneally and the other individuals kept in separate tanks were corresponding challenged with 1 ml of physiological water as the control. Fish were sacrificed in various times and three individual tissues were collected at each time. All animal experiments 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 the College of Marine science, Zhejiang Ocean University (number EC2015011).

Macrophage culture
For the macrophage isolation, the head kidneys from juvenile miiuy croaker were collected aseptically. Tissues were minced thoroughly with scissors and pushed carefully through a 100-m nylon mesh in L-15 medium containing penicillin (100 IU/ml), streptomycin (100 mg/ml), 2% fetal bovine serum (FBS), and heparin (20 units/ml) to give a single cell suspension. The filtered cell suspension was loaded onto 34/51% Percoll (Pharmacia, USA) density gradient, and then centrifuged at 400 ϫ g for 40 min at 4°C. Subsequently, the supernatant was removed and the cells at the interfaces were obtained with care and washed twice in L-15 medium at 300 ϫ g for 10 min at 4°C. Macrophages were cultured in L-15 containing 0.1% FBS at 26°C, 4% CO 2 . The next day, the cell pellet was re-suspended in fresh complete L-15 medium supplemented with 20% FBS.

RNA isolation and real-time quantitative PCR
Total RNA and small RNAs (Ͻ200 nucleotides) were isolated with TRIzol reagent (Invitrogen) and their miRcute miRNA Isolation Kit (Tiangen), respectively, following the manufacturer's protocol. Real-time quantitative PCR (qPCR) was performed on a 7300 real-time PCR system (Applied Biosystems, USA) as previously described (18). The relative expression level of mRNA was normalized by ␤-actin expression, whereas miRNA was normalized by 5.8S rRNA. All amplification reactions were carried out within a triplicate well of each sample and sequences of mRNA and miRNA primers listed in supplemental Table S1.

Plasmid construction and transfection
To construct the myd88 expression vector, the full-length CDS region and 3ЈUTR of the miiuy croaker myd88 gene was amplified by specific primer pairs harboring a FLAG tag and restricted endonuclease sites KpnI and EcoRI, and then inserted into pcDNA3.1 vector (Invitrogen). To construct the myd88-3ЈUTR reporter vector, the full-length 3Ј-UTR region of myd88 was amplified from cDNA of miiuy croaker. The PCR product was digested within NheI and SalI, respectively, which was then cloned into the pmirGLO luciferase reporter vector (Promega). The mutant-type of myd88-3ЈUTR reporter vector was conducted using Mut Express II Fast Mutagenesis Kit V2 (Vazyme) with specific primers (supplemental Table S1). Additionally, myd88-3ЈUTR or mutant-type was cloned into the pIZ/V5-His vector (Invitrogen), which contained the enhanced green fluorescent protein (GFP) sequence. To construct pre-miRNA vector, the pre-miR-214 sequence were amplified by PCR and then cloned into pcDNA6.2-GW/EmGFP vector (Invitrogen). All recombinant plasmids were extracted through the Endotoxin-free Plasmid DNA Miniprep Kit (Tiangen) and confirmed by Sanger sequencing before the Dual-Luciferase reporter assay. Before transient transfection, HEK293 cells were seeded in 24-well plates for 24 h. Cells were subsequently transfected with 100 ng of plasmids using Lipofectamine 2000 TM (Invitrogen), according to the manufacturer's instructions.

Prokaryotic expression and polyclonal antiserum
For prokaryotic expression, the full-length CDS region of miiuy croaker myd88 was cloned into EcoRI/XhoI sites of the pGEX-4T-1 vector (GE Healthcare) to construct the pGEX-4T-1-myd88 plasmid. Subsequently, the plasmid pGEX-4T-1-myd88 was transformed into the Escherichia coli BL21(DE3) strain and expressed as a protein containing myd88 fused with GST. The fusion protein was induced by isopropyl 1-thio-␤-Dgalactopyranoside and purified by GST-Bind Resin chromatography. The purified fusion protein was applied to immunize New Zealand White rabbits to raise a polyclonal anti-myd88 antiserum. Validation for polyclonal antiserum is documented in supplemental Fig. S1.

Dual-Luciferase reporter assays
For miRNA target identification, HEK293 cells were cotransfected with wild-type or mutant type myd88-3ЈUTR luciferase reporter vector, along with miR-214 mimics, inhibitors, and controls or pre-miR-214 plasmid. Additionally, HEK293 cells were cotransfected with the NF-B luciferase reporter plasmid, pRL-TK Renilla luciferase plasmid, myd88 expression plasmid, along with miR-214 mimics, inhibitors, and controls or pre-miR-214 plasmids for Dual-Luciferase reporter assay. After 24 or 48 h, the cells were collected and assayed for reporter activity using the Dual-Luciferase Reporter System (Promega) following the manufacturer's instructions and the relative luciferase activity value was achieved against the Renilla luciferase control. The results shown were done in triplicate for each experiment, and three independent experiments were conducted.

Western blotting
After 48 h post-transfection, total HEK293 cellular lysates or macrophages lysates were generated. The soluble protein concentrations were measured with a BCA Protein Assay Kit (Thermo Scientific), and an equal amount of protein was loaded for SDS-PAGE. Proteins were transferred onto PVDF membranes (Pall Corporation) in a semi-dry manner (Bio-Rad Trans Blot Turbo System). Membranes were blocked for 1 h with 5% dried skimmed milk powder in 100 mM TBST. Then, the membranes were incubated at 4°C overnight with anti-FLAG mouse monoclonal antibody (Beyotime) or polyclonal anti-myd88 antiserum and zebrafish ␤-actin monoclonal antibodies (Beyotime), respectively. The following day, the membranes were incubated with the secondary antibody conjugated with horseradish peroxidase at room temperature for 60 min (Beyotime). The immunoreactive proteins were detected using BeyoECL Plus (Beyotime) and digital imaging was performed with a cold CCD camera. The results shown were done in triplicate for each experiment, and three independent experiments were conducted. All of the results are from separate blots to avoid possible problems related to incomplete stripping.

Statistical analysis
All experiments were repeated three times. Data on relative gene expression was obtained using the 2 Ϫ⌬⌬CT method, and comparisons between groups were analyzed by one-way analysis of variance followed by Duncan's multiple comparison tests (19). All data were presented as the mean Ϯ S.E., significant differences between groups were determined by two-tailed Student's t test.
Author contributions-Q. C. and T. X. conceived and designed the experiments; Q. C., J. C., and T. X. performed the experiments: Q. C. and T. X. analyzed the data; Q. C., Y. S., J. C., and T. X. contributed reagents/materials/analysis tools; and Q. C., Y.S., J.C., and T. X. wrote the paper.