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

Qing Chu, Yuena Sun, Junxia Cui and Tianjun Xu
J Immunol April 15, 2017, 198 (8) 3274-3282; DOI: https://doi.org/10.4049/jimmunol.1602064

Abstract

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.

Introduction

Microbial pathogen recognition relies on several classes of germline-encoded pattern recognition receptors (PRRs), including TLRs, NOD-like receptors (NLRs), and RIG-I–like receptors, which then initiate host innate immune response and a series of inflammatory responses to defend against infectious pathogens. TLRs are the most studied PRRs and are important players involved in the regulation and orchestration of innate and adaptive immune systems (1, 2). Following recognition of corresponding ligands, TLRs activate multiple signaling pathways leading to the induction of genes involved in innate immunity. In most cases, TLRs mainly signal through the MyD88-dependent pathway and transduce signals through a series of cytoplasmic molecules, including MyD88, IL-1R–associated kinase (IRAK)1, IRAK4, and TNFR-associated factor (TRAF)6, which are critical for the activation of NF-κB transcription factors (3–6). Activation of NF-κB then promotes transcription of a series of genes, including IL-1β, IL-6, and TNF-α, for defense against invaders (7, 8). However, accumulating evidence shows that excessive TLR signaling activation will disrupt immune homeostasis, and furthermore it may induce the autoimmune and inflammatory diseases (9). As such, mechanisms for the negative regulation of TLR signaling have been intensively investigated.

To date, many negative regulators of TLR signaling have been identified and characterized (10). IRF4 competing with IRF5 to interact with MyD88 acts as a negative regulator of TLR signaling (11). Similarly, NLRX1 functioning as intracellular PRRs suppresses TLR signaling through interaction with TRAF6 and the IκB kinase (IKK) complex (12). Although many studies have focused on protein regulators, microRNAs (miRNAs) have emerged as important controllers of the innate immune response (13).

miRNA small noncoding RNAs consisting of ∼22 nt regulate gene expression through binding with the 3′ untranslated regions (UTRs) of target genes, resulting in protein translation repression or mRNA degradation (14, 15). miRNAs are implicated in the regulation of diverse biological processes, including proliferation and differentiation, cancer, apoptosis, and viral infections (16–18). As reported, as many as 60% of all mRNAs have been predicted to be regulated by miRNAs to some extent (19). miRNAs thereby serve as important transcription factors in controlling the protein content of a cell. Currently, a range of miRNAs has been widely studied in the regulation of TLR signaling pathways at different layers, including regulation of TLR expression, TLR-associated signaling proteins, and TLR-induced transcription factors and cytokines (13, 20). Compelling evidence shows that miRNAs in regulation of TLR downstream signaling molecules seem more effective than directly targeting TLRs (13). A recent study demonstrated that miRNA (miR)-146a, one of key TLR-induced miRNAs, inhibits the TLR signaling pathway by targeting IRAK1 and TRAF6 (21). Additionally, MyD88, a key downstream adaptor for most TLRs, has also been shown to be regulated by miRNAs, including miR-155 (22), miR-149 (23), and miR-203 (24). Overall, the evidence indicates that miRNA could result in timely and appropriate negative regulation of the TLR signaling pathway by targeting critical signaling proteins when a TLR is triggered.

In innate immune responses, the signal transduction of the TLR pathway is highly conserved from invertebrates to mammals (25). Moreover, MyD88 has been extensively studied in vertebrates; in comparison with other TLR adaptors, the structure of MyD88 is well conserved and its homolog in fish species may function similarly to the mammalian counterparts (26, 27). In zebrafish, MyD88 has been confirmed to be involved in the clearance of the bacterial infection via MyD88 knockdown in embryos (28). MyD88 in Atlantic salmon also has been implicated in regulation of the NF-κB promoter (29). However, the underlying mechanisms of miRNAs in regulation of the MyD88-depedent signaling pathway against varied pathogens or stimuli remain poorly understood in teleost fish. In this study, miR-3570 rapidly upregulated following stimulating with Vibrio anguillarum, the causative agent of vibriosis in cultivated fish, has been identified from miiuy croaker (Miichthys miiuy). Upregulated miR-3570 suppressed MyD88 expression, which subsequently repressed inflammatory cytokine genes through MyD88-mediated NF-κB signaling, thereby avoiding excessive inflammation. To the best of our knowledge, this report is the first to elucidate that miR-3570 is upregulated upon bacterial infection and acts as a negative regulator of MyD88-mediated signaling by targeting MyD88.

Materials and Methods

Sample and challenge

Healthy miiuy croakers (∼750 g) were obtained from Zhoushan Fisheries Research Institute (Zhejiang, China) and raised in aerated seawater tanks at 25°C for at least 1 wk. For the stimulation experiment, briefly, these healthy fishes were randomly divided into two groups in which the experimental individuals were challenged i.p. with 1 ml of V. anguillarum (1.5 × 108 CFU/ml) or a 1-ml suspension of LPS (1 mg/ml; Sigma-Aldrich), and the other individuals kept in separate tanks were correspondingly challenged with 1 ml of physiological water as the control. Fish were killed in various times, and the immune tissues (liver, spleen, kidney) were collected and then stored at −80°C for later use. 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 (approval no. EC2015011).

Cell culture and LPS exposure

HEK293 cells were cultured with DMEM high-glucose medium (HyClone) containing 10% FBS under condition with 5% CO2 at 37°C. The miiuy croaker macrophages were aseptically obtained from the head kidney of miiuy croakers as previously described (30) and cultured in L-15 medium supplemented with 20% FBS. For the LPS exposure, macrophages were challenged with 10 μg/ml LPS and harvested at different times for RNA extraction. Cells with no stimulation were collected as the control, and each experiment had three biological replicates.

RNA isolation and real-time quantitative PCR

Total RNA was isolated with TRIzol reagent (Invitrogen) following the manufacturer’s protocol. Real-time quantitative PCR (qPCR), using SYBR Premix Ex Taq (TaKaRa Bio), was performed on an ABI 7300 real-time PCR system (Applied Biosystems) as we previously described (30). Primers of genes were designed by Primer Premier 5 software and the data were normalized by the β-actin expression in each sample (Supplemental Table I).

For miRNA analysis, small RNA (<200 nt) was harvested using the miRcute miRNA isolation kit (Tiangen Biotech). The amplification reactions were carried out using the miRcute miRNA qPCR detection kit (Tiangen Biotech) with the following conditions: 95°C for 15 min, 40 cycles of two steps (95°C for 5 s, 60°C for 30 s) within triplicate well of each sample. Sequences of miRNA primers are listed in Supplemental Table I, and the relative expression level of miRNA was normalized by 5.8S rRNA expression.

Plasmid construction and transfection

To construct the MyD88 expression vector, the full-length coding sequence (CDS) region and 3′UTR of the miiuy croaker MyD88 gene was amplified by specific primer pairs with Flag tag and restricted endonuclease sites KpnI and EcoRI, and then inserted into pcDNA3.1 vector (Invitrogen). To construct a MyD88-3′UTR reporter vector, the full-length 3′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 pmirGLO luciferase reporter vector (Promega). The mutant-type of the MyD88-3′UTR reporter vector was conducted using a Mut Express II fast mutagenesis kit v2 (Vazyme Biotech) with specific primers (Supplemental Table I). Additionally, MyD88-3′UTR or the mutant-type was cloned into the pIZ/V5-His vector (Invitrogen), which contained the sequence of enhanced GFP. To construct the premiRNA vector, the premiR-3570 sequences were amplified by PCR and then cloned into pcDNA6.2-GW/EmGFP vector (Invitrogen). All recombinant plasmids were extracted through an endotoxin-free plasmid DNA miniprep kit (Tiangen Biotech) 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 (Invitrogen), according to the manufacturer’s instructions. For each transfection experiment, plasmid encoding enhanced GFP (EGFP) was transfected as the positive control to verify transfection efficiency.

miRNA mimics and inhibitors

miR-3570 mimic (dsRNA oligonucleotides), miR-3570 inhibitor (single-stranded oligonucleotides chemically modified by 2′-Ome), and control oligonucleotides were commercially synthesized by GenePharma (Shanghai, China). Their sequences are as follows: miR-3570 mimic, sense, 5′-UACAAUCAACGGUCGAUGGUUU-3′, antisense, 5′-ACCAUCGACCGUUGAUUGUAUU-3′; mimics control, sense, 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense, 5′-ACGUGACACGUUCGGAGAATT-3′; miR-3570 inhibitor, 5′-AAACCAUCGACCGUUGAUUGUA-3′; and inhibitors control, 5′-CAGUACUUUUGUGUAGUACAA-3′. HEK293 cells or macrophages were seeded into 24-well plates and incubated overnight. Cells were subsequently transfected with 30–100 nM of each oligonucleotide 24 h before LPS stimulation using Lipofectamine 2000 (Invitrogen).

RNA interference

The MyD88-specific small interfering RNA (siRNA) sequences were 5′-GCUCGAAACAAACGCCUUATT-3′ (sense) and 5′-UAAGGCGUUUGUUUCGAGCTT-3′ (antisense). The scrambled control RNA sequences were 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense). The MyD88-specific siRNA transfection was performed with Lipofectamine 2000 (Invitrogen). Macrophages were seeded into 24-well plates and incubated overnight. Cells were subsequently transfected with 100 nM of each siRNA for 24 h before LPS stimulation.

Prokaryotic expression and polyclonal antiserum

For prokaryotic expression, the full-length CDS region of miiuy croaker MyD88 was cloned into EcoRI/XhoI sites of pGEX-4T-1 vector (GE) to construct pGEX-4T-1-MyD88 plasmid. Subsequently, the plasmid pGEX-4T-1-MyD88 was transformed into the BL21 (DE3) Escherichia coli strain and expressed as a protein containing MyD88 fused with GST. The fusion protein was induced by isopropyl β-d-thiogalactoside 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.

Dual-Luciferase reporter assays

For miRNA target identification, HEK293 cells were cotransfected with wild-type or mutant type MyD88-3′UTR luciferase reporter vector, along with miR-3570 mimics, inhibitors, and controls or premiR-3570 plasmid. Additionally, HEK293 cells were cotransfected with NF-κB, IFN-stimulated response element (ISRE), IFN-α, IFN-β, or IFN-γ luciferase reporter plasmid, pRL-TK Renilla luciferase plasmid, MyD88 expression plasmid, along with either miR-3570 mimics, inhibitors, and controls or premiR-3570 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 posttransfection, total HEK293 cellular lysates or macrophages lysates were generated using 1× SDS-PAGE loading buffer. Then, SDS-PAGE was performed within equal amounts of protein, followed by protein transfer onto polyvinylidene difluoride membranes (Pall) in a semidry manner (Trans-Blot Turbo system; Bio-Rad Laboratories). 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 mAb (Beyotime) or polyclonal anti-MyD88 antiserum and zebrafish β-actin mAbs (Beyotime). The following day, the membranes were incubated with the secondary Ab conjugated with HRP at room temperature for 60 min (Beyotime). The immunoreactive proteins were detected by using BeyoECL Plus (Beyotime), and digital imaging was performed with a cold CCD camera.

Statistical analysis

Data on relative gene expression were obtained using the 2−∆∆CT method, and comparisons between groups were analyzed by one-way ANOVA followed by a Duncan multiple comparison test (31). All data are presented as the mean ± SE; significant differences between groups were determined by a two-tailed Student t test.

Results

V. anguillarum infection significantly upregulated miR-3570 expression

Our previous study proposed that miR-3570 in miiuy croaker could be increased by V. anguillarum infection (32). To better explore the expression of miR-3570 upon pathogen stimulation, we investigated the levels of miR-3570 in V. anguillarum–infected miiuy croaker liver and spleen samples using quantitative RT-PCR. The results showed that miR-3570 was rapidly upregulated and reached a peak at 12 h and then kept the rising trend after V. anguillarum infection in liver samples (Fig. 1A). Consistent with this, the expression profiles of miR-3570 in spleen samples were detected and showed significant time-dependent upregulation (Fig. 1B). To confirm the above results, purified LPS, the endotoxin of Gram-negative bacteria, was conducted as pathogen to examine miR-3570 expression in vivo and in vitro. Data revealed that LPS also exhibited an activating effect on miR-3570 expression in both LPS exposure macrophages and LPS-injected immune tissue samples (Fig. 1C–F). Thus, our findings strongly demonstrate that miR-3570 expression could be upregulated by V. anguillarum infection, as well as LPS, and may be involved in the regulation of the immune response.

FIGURE 1.
FIGURE 1.

The expression profiles of miR-3570 detected by qPCR. Expression profiles of miR-3570 after V. anguillarum infection in liver (A) and spleen (B) tissue samples are shown. (C) Expression profile of miR-3570 after LPS stimulation in macrophages. Expression profiles of miR-3570 after LPS injection in liver (D), spleen (E), and kidney tissues (F) are shown. The data were normalized to 5.8S rRNA. Results are standardized to 1 in control cells. Data represent the mean ± SE from three independent experiments performed in triplicate.

miR-3570 inhibits the production of inflammatory cytokines in LPS-stimulated macrophages

To confirm the role of miR-3570 in the bacteria-induced inflammatory response, we then investigated miR-3570 on the regulation of inflammatory cytokine production after LPS stimulation. First, the effects of synthetic miR-3570 mimic and inhibitor on the expression of miR-3570 were assessed. Given that the abundance of the physiological miRNAs can be changed via miRNA mimic and inhibitor, of which miRNAs mimic could simulate naturally occurring mature miRNAs and the inhibitor blocks the activity of endogenous miRNAs by complementarity, the macrophages were transfected with miR-3570 mimic and miR-3570 inhibitor, respectively. As expected, the miR-3570 mimic increased miR-3570 expression whereas the miR-3570 inhibitor decreased miR-3570 expression (Fig. 2A). We then probed whether LPS stimulation leads to induction of inflammatory cytokine gene expression in macrophages, and we found that certain inflammatory mediators, including NF-κB, TNF-α, IL-6, and IL-1β, were rapidly induced after LPS stimulation, reaching peak concentrations within 6 or 12 h (Fig. 2B).

FIGURE 2.
FIGURE 2.

miR-3570 suppresses the mRNA expression of NF-κB, TNF-α, IL-6, and IL-1β in LPS-stimulated macrophages. (A) Miiuy croaker macrophages were transfected with miR-control (Ctrl) or miR-3570 (left) and Ctrl inhibitor or miR-3570 inhibitor (right) within a final concentration of 100 nM. At 48 h posttransfection, miR-3570 expression was measured by qPCR and normalized to 5.8S rRNA. Results are standardized to 1 in Ctrl cells. (B) The macrophages were stimulated with or without LPS in different times. The expressions of NF-κB, TNF-α, IL-6, and IL-1β mRNA levels were determined by qPCR and normalized to the expression of β-actin in each sample. (C) The macrophages were transfected with miR-3570 mimic or miR-Ctrl. After 24 h, the cells were stimulated with LPS and the expression levels of the above genes were analyzed by qPCR after another 24 h. (D) The macrophages were transfected with miR-3570 inhibitor or Ctrl inhibitor. After 24 h, the cells were stimulated with LPS and the expression levels of the above genes were analyzed by qPCR after another 24 h. Data are presented as the mean ± SE from three independent experiments performed in triplicate. **p < 0.01, *p < 0.05 versus the Ctrls.

Next, we sought to explore the role of miR-3570 in modulating inflammatory mediator expression against LPS. The macrophages were then transfected with control miRNA, miR-3570, miR-3570 inhibitor, or control inhibitor prior to LPS exposure for up to 24 h. The results showed that after time-dependent LPS stimulation, miR-3570 overexpression markedly decreased the expression of NF-κB, TNF-α, IL-6, and IL-1β compared with control miRNA (Fig. 2C). In contrast, increases in NF-κB, TNF-α, IL-6, and IL-1β expression were observed with the introduction of miR-3570 inhibitor after LPS stimulation (Fig. 2D) compared with control inhibitor. These data demonstrate that miR-3570 could suppress the production of certain inflammatory mediators, including NF-κB, TNF-α, IL-6, and IL-1β, which may play a negative role in response to LPS stimulation in miiuy croaker.

miR-3570 regulates the expression of components of the MyD88-depedent signaling cascade

In mammals, TLR4 recognizes LPS and mediates the MyD88-dependent pathway (25). Although TLR4 in fish is lost or fails to recognize LPS (33), other TLRs may substitute for this function because TLRs in fish display high variety and distinct features. There is compelling evidence that the signal transduction of the TLR pathway is highly conserved from invertebrates to mammals. MyD88, the principal transducer of TLR signaling, is also well conserved, and its homolog in fish species may function similarly to the mammalian counterparts (27, 28). Therefore, we evaluated proteins in the MyD88-depedent signaling cascade to identify potential targets of miR-3570. As shown in Fig. 3A–C, the expressions of both MyD88-interacting molecules (IRAK1 and IRAK4) and TRAF6 were decreased by miR-3570 overexpression, whereas they were increased by miR-3570 inhibitor treatment in comparison with the control group. Subsequently, we further examined the mRNA expression of MyD88 in the same experimental samples using qPCR, and we found that MyD88 mRNA levels showed a similar trend compared with the IRAK4/IRAK1/TRAF6 complex after transfection with miR-3570 mimic (Fig. 3D) and inhibitor (Fig. 3F). In agreement with this, Western blot analysis of MyD88 revealed that the protein abundance of MyD88 in macrophages could be decreased upon dose-dependent miR-3570 overexpression (Fig. 3E) and increased upon dose-dependent miR-3570 inhibition (Fig. 3G). These results show that miR-3570 attenuates the expression of genes involved in theMyD88-mediated signaling cascade and MyD88 may be a potential target of miR-3570.

FIGURE 3.
FIGURE 3.

miR-3570 regulates the expression of MyD88, IRAK1, IRAK4, and TRAF6. The miiuy croaker macrophages were transfected with miR-3570 or miR-control (Ctrl) and miR-3570 inhibitor or Ctrl inhibitor (final concentration, 100 nM). After 48 h, IRAK1 (A), IRAK4 (B), TRAF6 (C), and MyD88 (D and F) mRNA levels were determined by qPCR and normalized to β-actin. Results are standardized to 1 in Ctrl cells. MyD88 protein levels were determined by Western blot and normalized to β-actin (E and G). Data are presented as the mean ± SE from three independent triplicated experiments. *p < 0.05 versus the Ctrls.

MyD88 is the target of miR-3570

To verify the potential target of miR-3570, we next used TargetSan (34) software to predict the putative miR-3570 binding site in the 3′UTR of MyD88 (Fig. 4A). Reporter plasmid was constructed by cloning miiuy croaker MyD88-3′UTR into the pmirGLO luciferase reporter vector within the mutation at the miR-3570 binding site as control. Additionally, the predicted premiR-3570 sequence of miiuy croaker was cloned into the pcDNA6.2-GW/EmGFP vector to constructed premiR-3570 plasmid (Fig. 4A). After 48 h, we observed that both miR-3570 mimic and premiR-3570 plasmid were sufficient to decrease luciferase activity when cotransfected with MyD88-3′UTR reporter plasmid into HEK293 cells, whereas the mutant-type led to a complete abrogation of the negative effect (Fig. 4B).

FIGURE 4.
FIGURE 4.

miR-3570 targets miiuy croaker MyD88. (A) Sequence alignment of miR-3570 and premiR-3570 and its construction plasmids. (B) HEK293 cells were cotransfected with pmirGLO empty plasmid, the wild-type of MyD88-3′UTR (WT), and the mutant type of MyD88-3′UTR (MT), together with miR-3570 or miR-control (Ctrl) and premiR-3570 or its Ctrl. After 48 h, luciferase activity was determined and normalized to Renilla luciferase activity. (C) HEK293 cells were cotransfected with MyD88-3′UTR (WT), together with miR-3570 or miR-Ctrl and miR-3570 inhibitor or Ctrl inhibitor. After 48 h, luciferase activity was determined and normalized to Renilla luciferase activity. (D) HEK293 cells were cotransfected with MyD88-3′UTR (WT), together with miR-3570 (30–100 nM) (left) or premiR-3570 (10–100 ng) (right) in a concentration gradient manner for 48 h. A Ctrl miRNA or pcDNA6.2 was used as Ctrl. The luciferase activity value was achieved against the Renilla luciferase activity. (E and F) HEK293 cells were cotransfected with pIZ/EGFP-MyD88-3′UTR or empty vector, together with miR-3570 or miR-Ctrl (E) and premiR-3570 or pcDNA6.2 (F). At 48 h posttransfection, the fluorescence intensity was evaluated (original magnification ×100). Data are presented as the mean ± SE from three independent experiments performed in triplicate. *p < 0.05 versus the Ctrls. MT, mutant; WT, wild-type.

The significant downregulation mechanism was further investigated using miR-3570 mimic and inhibitor. We transfected MyD88-3′UTR reporter plasmid, together with equal amount of oligonucleotides, into HEK293 cells. As shown in Fig. 4C, transfection with miR-3570 mimic itself decreased the luciferase activity, whereas the downregulation was inhibited when cells were cotransfected with equal amounts of miR-3570 mimic and miR-3570 inhibitor. Additionally, a dose-dependent effect of the miR-3570 mimic and premiR-3570 plasmid on inhibition luciferase activity could be observed (Fig. 4D). Moreover, both miR-3570 mimic and premiR-3570 plasmid could downregulate GFP gene expression when the MyD88-3′UTR was cloned into the pIZ/EGFP vector in HEK293 cells (Fig. 4E, 4F). Parallel to these results, GFP gene expression could not be regulated with a mutant-type of MyD88-3′UTR (Fig. 4E, 4F). Collectively, the data sufficiently demonstrate that miR-3570 targets the 3′UTR of MyD88.

miR-3570 inhibits the expression of MyD88 at the posttranscriptional level

To determine miR-3570 function in regulation of MyD88 expression, we next sought to cotransfect miR-3570, together with MyD88 expression plasmid into HEK293 cells. To construct MyD88 expression plasmid, the full-length CDS region and the 3′UTR of the miiuy croaker MyD88 gene were amplified by specific primer pairs and cloned into pcDNA3.1 vector with Flag tag. As shown in Fig. 5A, miR-3570 decreased MyD88 expression in both protein and mRNA levels. Additionally, the results further examined by cotransfecting with premiR-3570 revealed that premiR-3570 also decreased MyD88 expression in a dose-dependent manner (Fig. 5B). These data, together with results shown in Fig. 3D–G, indicated that MyD88 expression could be inhibited by miR-3570 via both translational inhibition and mRNA degradation.

FIGURE 5.
FIGURE 5.

miR-3570 is a negative regulator of MyD88. (A) HEK293 cells were cotransfected with MyD88 expression plasmid, together with miR-3570 or miR-control (Ctrl). After 48 h, MyD88 protein levels were determined by Western blot and normalized to β-actin (left); MyD88 mRNA levels were determined by qPCR and normalized to β-actin (right). To construct MyD88 expression plasmid, the full-length CDS region and 3′UTR of miiuy croaker MyD88 gene were amplified by specific primer pairs with Flag tag. (B) HEK293 cells were cotransfected with MyD88 expression plasmid, together with premiR-3570 or empty vector with concentrate gradient. After 48 h, MyD88 protein levels were determined by Western blot and normalized to β-actin (left); MyD88 mRNA levels were determined by qPCR and normalized to β-actin (right). (C and D) Miiuy croaker macrophages were cotansfected with miR-3570 or miR-Ctrl (C) and miR-3570 inhibitor or Ctrl-inhibitor (D). After 24 h, cells were stimulated with or without LPS. The expression of MyD88 mRNA was subsequently analyzed at 3, 6, and 12 h poststimulation by qPCR. Data are presented as the mean ± SE from three independent experiments performed in triplicate. *p < 0.05 versus the Ctrls.

Additionally, given that MyD88 could be upregulated by LPS stimulation, we further examined miR-3570 in the regulation of MyD88 expression after macrophage treatment with LPS. As shown in Fig. 5C and 5D, miR-3570 mimic decreased mRNA expression of MyD88, whereas the inhibitor increased its expression after LPS exposure. These data revealed that miR-3570 targets and negatively regulates MyD88 expression levels after LPS stimulation in miiuy croaker.

miR-3570 suppresses the MyD88-mediated NF-κB pathway

Prior studies have demonstrated that the MyD88-dependent pathway results in activation of NF-κB and MAPKs as well as transcription of several inflammatory genes (3). Reports about zebrafish MyD88 also indicated that overexpression of zebrafish MyD88 in HEK293T cells could activate both NF-κB and IFN-β promoters (35). To determine the regulation role of miiuy croaker MyD88, we test whether overexpression of MyD88 affects the activation of NF-κB, IFN-α, IFN-β, IFN-γ, and ISRE. As shown in the Dual-Luciferase reporter assays (Fig. 6A), overexpression of MyD88 in HEK293 cells was sufficient to activate NF-κB reporter gene, whereas it showed no obvious effect on IFN-α, IFN-β, IFN-γ, and ISRE. Given that miR-3570 targets and negatively regulates the expression of MyD88, we then examined whether overexpression of miR-3570 inhibits the activation of NF-κB reporter plasmid. To this end, we transfected MyD88 expression plasmid, together with miR-3570 mimic and miR-3570 inhibitor, into HEK293 cells, and each assay was transfected within equal amounts of oligonucleotides. As shown in Fig. 6B, at 48 h posttransfection, miR-3570 mimic significantly attenuated the activation of NF-κB induced by overexpression of MyD88. However, when we cotransfected with the same amounts of miR-3570 mimic and miR-3570 inhibitor into HEK293 cells, the negative effect on NF-κB was remarkably weakened. To better demonstrate the negative role of miR-3570 in MyD88 mediated for NF-κB activation, a concentration and time gradient experiment was conducted. As shown in Fig. 6C, both miR-3570 mimic and premiR-3570 plasmid suppress the activation of NF-κB in a dose-dependent manner. Moreover, in comparison with 48 h posttransfection, the negative regulation of both miR-3570 mimic and premiR-3570 plasmid showed more efficiency at 24 h posttransfection (Fig. 6D). In line with the above findings, a signaling model has been performed (Fig. 6E). Taken together, these data demonstrate that miR-3570 negatively regulates the MyD88-mediated NF-κB pathway in a manner that depends on targeting MyD88.

FIGURE 6.
FIGURE 6.

Overexpression of miR-3570 suppresses the MyD88-mediated NF-κB pathway. (A) HEK293 cells were cotransfectd with MyD88 expression plasmid, pRL-TK Renilla luciferase plasmid, together with luciferase reporter constructs driven by NF-κB, IFN-α, IFN-β, IFN-γ, and ISRE reporter genes. After 24 h, the luciferase activity was measured, and results are presented relative to the luciferase activity in control (Ctrl) cell. (B) miR-3570 or miR-Ctrl and miR-3570 inhibitor or Ctrl inhibitor were cotransfected with MyD88 expression plasmid, pRL-TK Renilla luciferase plasmid, together with luciferase reporter NF-κB into HEK293 cells. After 48 h, the luciferase activity was measured, and results are presented relative to the luciferase activity in Ctrl cells. (C) The concentration gradient experiment of miR-3570 (left) or premiR-3570 (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-3570 (left) or premiR-3570 (right). (E) The proposed model for miR-3570 inhibition of inflammatory cytokine secretion via MyD88. Data are presented as the mean ± SE from three independent experiments performed in triplicate. **p < 0.01, *p < 0.05 versus the Ctrls.

MyD88 siRNA decreases the production of inflammatory cytokines in LPS exposure macrophages

To confirm the contribution of MyD88 on regulation of inflammatory cytokines, the macrophages were transfected with MyD88-specific siRNA. siRNAs effectively inhibited MyD88 expression in macrophages (Fig. 7A). Knockdown of MyD88 significantly decreased the levels of NF-κB expression after LPS stimulation in macrophages (Fig. 7B), which produced effects similar to those of miR-3570 overexpression. Similar downregulation trends were also detected in other IL-1β, IL-6, and TNF-α (Fig. 7C–E).

FIGURE 7.
FIGURE 7.

The mRNA expression levels of MyD88 and inflammatory cytokines after MyD88 interference. (A) Miiuy croker macrophages were transfected with control siRNA (si-Ctrl) or siRNA against MyD88 (si-MyD88). After 48 h, MyD88 mRNA levels were determined by qPCR and normalized to β-actin (left); MyD88 protein levels were determined by Western blot and normalized to β-actin (right). (B–E) After 24 h transfection with si-Ctrl or si-MyD88, macrophages were then stimulated with LPS for another 24 h and the expression of NF-κB (B), TNF-α (C), IL-6 (D), and IL-1β (E) were determined. Data are presented as the mean ± SE from three independent experiments performed in triplicate. **p < 0.01, *p < 0.05 versus the controls.

Discussion

miRNAs are involved in the regulation of multiple biological processes via modulating the expression of target genes. Recent studies have indicated that these molecules also play important roles in the immune system (36). The role of miRNAs in the bacteria-triggered inflammatory response has not been investigated in detail, especially in fish species. V. anguillarum, a curved rod type of Gram-negative bacterium, results in high mortality and severe epidemic vibriosis with symptoms of hemorrhage septicemia in many marine animals (37, 38). In this study, we found that the expression of miR-3570 was rapidly and constantly upregulated in miiuy croaker after stimulation with V. anguillarum and LPS. Upregulated miR-3570 could suppress the production of bacteria-triggered inflammatory cytokines. miR-3570 also attenuated the expression of genes involved in the MyD88-mediated signaling cascade by targeting MyD88, therefore negatively regulating the NF-κB pathway. Collectively, these data revealed that miR-3570 decreases the production of inflammatory cytokines in LPS-stimulated macrophages through negatively regulating the NF-κB signaling pathway by modulating MyD88. This could be a novel finding on the negative regulation of the NF-κB pathway, which also suggests a novel mechanism for avoiding excessive inflammation in teleost fish, as well as in mammals.

NF-κB is the central transcription factor that directly regulates the encoding of a variety of important genes, including inflammatory cytokines, and is involved in inflammation and the immune response (39). Because of the importance of NF-κB, various cellular signal transduction adaptors tightly cooperate to regulate the NF-κB pathway. The excessive and prolonged production of proinflammatory mediators largely depended on the NF-κB pathway, such as TNF-α and IL-6, and may result in tissue damage (40). In mammals, several studies reported the involvement of miRNAs in the negative regulation of the NF-κB pathway. In the epithelial cell line, miR-155 could inhibit the production of proinflammatory cytokines through the activation of NF-κB by targeting IKKε (41). Similarly, miR-7578 identified in epididymis could inhibit the inflammatory response through NF-κB signaling, and this inhibition is accomplished through the miR-7578 target gene Egr1 (42). However, little is known about miR-3570 in the regulation of the NF-κB pathway. This study demonstrated that upon V. anguillarum infection, miR-3570 is immediately induced. Inducible miR-3570 downregulates the NF-κB signaling pathway by suppressing MyD88 in LPS-stimulated macrophages, which then inhibits the excessive inflammatory responses. These results provide new insight into the negative regulation of miR-3570 in the NF-κB pathway.

TLR-mediated signaling, which is highly conserved in vertebrates, represents the canonical mechanism of NF-κB activation (43, 44). After recognizing the PAMPs, TLRs bind adaptor proteins, such as MyD88, and then transduce the signals to activate of NF-κB, which triggers proinflammatory or innate immune responses (5, 6). To avoid overactivation or insufficient activation of NF-κB, the process is tightly regulated by a battery of endogenous mechanisms. For instance, NLRX1 has been reported to negatively regulate TLR-induced NF-κB activation by dynamically interacting with TRAF6 and the IKK complex (12). Moreover, a number of miRNAs, which result in timely and appropriate regulation of target genes, have been indicated to participate in the regulation of the NF-κB pathway through modulating the activities of many cellular adaptors, and these miRNAs include miR-31, miR-223, miR-195, and others (45–47).

With the exception of TLR3, all mammalian TLRs use MyD88 to commence signaling. Thus, it may be the most effective to directly regulate MyD88 in host immune response against invaders. Accumulating studies have suggested that MyD88 was tightly regulated during the inflammatory response. In mammals, each highly conserved miRNA probably targets several hundred distinct mRNAs, and each gene could be regulated by many miRNAs. With regard to MyD88, an array of miRNAs has been shown to regulate MyD88 in mammals, including miR-155, miR-149, and miR-203 (22–24). However, it is not known whether miRNAs have a similar role in fish species. In our study, miR-3570 was first demonstrated to modulate and inhibit the expression of MyD88; meanwhile, it was also indicated to negatively regulate the NF-κB pathway.

In summary, the present study demonstrated that miR-3570 inhibits the inflammatory response through negatively regulating the NF-κB pathway. The negative regulation mechanism is responsible for the inflammatory reaction after Gram-negative bacteria infection. Collectively, our findings not only expand the knowledge of regulatory mechanisms of the innate signaling pathway in teleost fish, but they also provide new insights into the regulatory mechanism of the innate signaling pathway in mammals.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Natural Science Foundation of China Grants 31370049 and 31672682 and by Natural Science Foundation of Zhejiang Province Grant LR14C040001.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CDS
    coding sequence
    EGFP
    enhanced GFP
    IKK
    IκB kinase
    IRAK
    IL-1R–associated kinase
    ISRE
    IFN-stimulated response element
    miR/miRNA
    microRNA
    NLR
    NOD-like receptor
    PRR
    pattern recognition receptor
    qPCR
    real-time quantitative PCR
    siRNA
    small interfering RNA
    TRAF
    TNFR-associated factor
    UTR
    untranslated region.

  • Received December 7, 2016.
  • Accepted February 2, 2017.

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