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RESEARCH ARTICLE | SEPTEMBER 15 2020
Inducible MicroRNA-217 Inhibits NF-κB– and IRF3-Driven Immune
Responses in Lower Vertebrates through Targeting TAK1 
Lei Zhang; ... et. al
J Immunol (2020) 205 (6): 1620–1632.
https://doi.org/10.4049/jimmunol.2000341

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 The Journal of Immunology

Inducible MicroRNA-217 Inhibits NF-kB– and IRF3-Driven

Immune Responses in Lower Vertebrates through
Targeting TAK1

Lei Zhang,*,1 Qing Chu,*,†,1 Renjie Chang,*,‡,x and Tianjun Xu*,†,‡,x

Upon recognition of bacterial or viral components by pattern recognition receptors, cells could be activated to produce inflam-
matory cytokines, type I IFN, and IFN-stimulated genes. These antibacterial and antiviral immunities are tightly regulated by the
host to prevent inappropriate immune responses. MicroRNAs (miRNAs) have emerged as an essential regulatory network with
profound effects on mammalian inflammation and immune responses, but the regulatory networks of miRNA-mediated immune

Downloaded from http://journals.aai.org/jimmunol/article-pdf/205/6/1620/1459609/ji2000341.pdf by guest on 17 June 2024

response in lower vertebrates remain largely unknown. In this study, we report a miRNA, miR-217, identified from miiuy croaker,
which plays a negative role in host antiviral and antibacterial immunity. We found that miR-217 could be abundantly expressed
upon Gram-negative bacteria, as well as rhabdovirus infection. Inducible miR-217 suppresses the production of inflammatory
cytokines and type I IFN by targeting TAK1, thereby avoiding excessive inflammation. Particularly, we revealed that miR-217
modulates the antibacterial and antiviral immunity through TAK1-mediated NF-kB and IRF3 signaling pathways. The collective
results indicate that miR-217 acts as a negative feedback regulator involved in host antibacterial and antiviral immune responses,
which will provide insights into the intricate networks of host–virus interaction in lower vertebrates. The Journal of Immunol-
ogy, 2020, 205: 1620–1632.

M
icrobial infection triggers complex interactions be- sensors for DNA), in which TLRs and RLRs are the main
tween the host and the pathogen. Host–pathogen in- PRRs (2, 4).
teractions are generally initiated through recognition TLRs are the most widely studied PRRs and are considered as
of conserved signature molecular structures termed as pathogen- the primary sensors of pathogens (1, 5). Upon ligand binding,
associated molecular patterns (PAMPs). PAMPs are sensed by TLRs use Myd88 or TRIF as adaptor proteins, which recruit and
host’s pathogen recognition receptors (PRRs), which are activate a series of signaling molecules, such as IRAK family
evolutionarily conserved and germline-encoded host sensors members, TNFR-associated factor 6 (TRAF6), and TGF-b–acti-
(1, 2). Upon pathogen recognition, PRRs rapidly initiate a vated kinase 1 (TAK1) (6). TRAF6 functions together with a di-
battery of immune immunity through the induction of a va- meric ubiquitin conjugating enzyme complex Ubc13-Uev1A to
riety of inflammatory cytokines, chemokines, and type I IFNs catalyze the synthesis of Lys63-linked polyubiquitin chains that
(3). PRRs are evolutionally conserved receptors that con- result in the activation of a protein kinase complex containing
sists of several classes, including TLRs, RIG-I–like receptors TAK1, TAB1, and TAB2 (6–9). The activated TAK1 then phos-
(RLRs), NOD-like receptors, and DNA receptors (cytosolic phorylates the IkB kinase (IKK) complex, leading to the activation
of transcription factors AP-1 and nuclear translocation of NF-kB
*Laboratory of Fish Molecular Immunology, College of Fisheries and Life Science,
(8, 9). Meanwhile, the IRF family is also activated via TBK1/
Shanghai Ocean University, Shanghai 201306, China; †Laboratory of Marine Biology IKKε. Different from the TLRs-mediated antiviral defense, RLRs
and Biotechnology, Qingdao National Laboratory for Marine Science and Technol- function as cytoplasmic sensors for viral RNA detection. Upon
ogy, Qingdao 266200, China; ‡Key Laboratory of Exploration and Utilization of
Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, effective recognition, RLRs recruit of the mitochondrial antiviral
Shanghai 201306, China; and xNational Pathogen Collection Center for Aquatic signaling protein (MAVS; also known as VISA, IPS-1, or CAR-
Animals, Shanghai Ocean University, Shanghai 201306, China DIF) (10–12). MAVS contains multiple TRAF-interacting motifs,
1
L.Z. and Q.C. contributed equally. which interacts with TRAF family members to signal the tran-
ORCID: 0000-0003-3606-8069 (T.X.). scription of type I IFN and inflammatory cytokines. Similar to the
Received for publication March 27, 2020. Accepted for publication July 11, 2020. TLR signaling, TAK1 plays a pivotal role in NF-kB activation,
This work was supported by the National Natural Science Foundation of and IRF3 is directly activated by TBK1/IKKε (11, 12). Notably,
China (31822057) and the National Key Research and Development Project NF-kB activation is an important common event in both TLR and
(2018YFD0900503).
RLR signaling pathways.
Address correspondence and reprint requests to Dr. Tianjun Xu, Shanghai Ocean
University, Shanghai 201306, China. E-mail address: tianjunxu@163.com TAK1 is a member of the MAPK kinase kinase (MAPKKK)
The online version of this article contains supplemental material.
family and was first identified in the TGF-b signaling pathway
(13). TAK1 is evolutionally conserved, and it has been shown to
Abbreviations used in this article: EPC, epithelioma papulosum cyprinid; IKK, IkB
kinase; ISG, IFN-stimulated gene; MAVS, mitochondrial antiviral signaling protein; function as an upstream activator of NF-kB and MAKPs in IL-1R
miRNA, microRNA; MKC, miiuy croaker kidney cell line; MOI, multiplicity of signaling pathways. Furthermore, accumulating evidences have
infection; PAMP, pathogen-associated molecular pattern; PRR, pathogen recognition
receptor; RLR, RIG-I–like receptor; SCRV, Siniperca chuatsi rhabdovirus; si-TAK1, reported that TAK1 could be activated by TNF, bacterial LPS, and
TAK1-specific small interfering RNA; TAK1, TGF-b–activated kinase 1; TRAF6, latent membrane protein 1 from EBV (14–17), which indicate that
TNFR-associated factor 6; TSS, transcription start site; 39-UTR, 39-untranslated TAK1 is critical for both antibacterial and antiviral innate im-
region.
munity. More recently, researches have shown that TAK1 could
Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 also induce the activation of TBK1-IRF3 and IRF3-dependent

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000341
The Journal of Immunology 1621

gene expressions (18). TAK1 is constitutively activated by human saline was used to challenge the individuals. Afterwards, fishes were re-
T cell lymphotropic virus 1 (HTLV-1) Tax oncoprotein, and Tax- spectively sacrificed at different time points, and the spleen tissues were
collected for RNA extraction. All animal experimental procedures were
induced TAK1 activation induces TBK1-IRF3 activation and the performed in accordance with the National Institutes of Health’s Guide for
expression of several IFN-inducible genes (18, 19). As a pivotal the Care and Use of Laboratory Animals, and the experimental protocols
kinase, the mechanisms and regulations of TAK1-mediated sig- were approved by the Research Ethics Committee of Shanghai Ocean
naling have been extensively studied. Growing evidence has University (No. SHOU-DW-2018-047).
shown that a series of host factors are implicated in regulating Cell culture
TAK1-mediated signaling. For instance, ubiquitin-specific pepti-
Miiuy croaker kidney cell lines (MKC) were cultured in L-15 medium
dase 4 (USP4) have been identified to targets TAK1 and thus
(HyClone) supplemented with 15% FBS (Life Technologies). Epithelioma
negatively regulate TNF-a–induced NF-kB activation (20). It has papulosum cyprinid (EPC) cells were maintained in medium 199 (Invitrogen)
been reported that ATP-binding cassette, subfamily B (TAP1) supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml strepto-
plays a novel role in the negative regulation of virus-triggered mycin at 28˚C in 5% CO2. Miiuy croaker macrophages were isolated from
NF-kB signaling by targeting the TAK1 complex (21). Compared head kidney samples as described (30). In brief, tissues were aseptically
pushed through a 100-mm nylon mesh to give cell suspension which was
with mammalian TAK1, however, the functions and mechanisms of then loaded onto 34%/51% Percoll (Pharmacia) density gradient to ob-
TAK1 in fish are poorly understood. tain macrophages. The cells were cultured in L-15 (HyClone) medium
Recently, an increasing number of studies has highlighted the supplemented with 20% FBS and seeded into six-well plates at 26˚C in
function of microRNAs (miRNAs) in regulating innate immune 4% CO2.

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pathways. miRNAs are small noncoding RNAs (∼19–23 nucleo- Plasmid construction
tide) emerging as translational repressors of gene transcripts by
To construct TAK1 expression plasmid, the full-length coding sequence and
binding to their 39-untranslated regions (39-UTRs), resulting in 39-UTR of miiuy croaker TAK1 gene were amplified by PCR and inserted
target transcripts degradation or protein translation inhibition (22, into pcDNA3.1 vector with a Flag tag. At the same time, the TAK1-39UTR
23). Accumulating evidences on the functions of miRNAs have sequence of M. miiuy, Sciaenops ocellatus, Larimichthys crocea, or Danio
shown the correlation between miRNAs and PRRs signaling. For rerio were inserted into the pmir-GLO vector to construct the wild-type
example, miR-146 is involved in the regulation of MyD88- TAK1-39UTR plasmid, respectively. The mutant-types of TBK1 39-UTR
reporter were constructed by using Mut Express II Fast Mutagenesis Kit
dependent signaling pathway by targeting IRAK1 and TRAF6 V2 (Vazyme) with specific primers (Supplemental Table I). Moreover, the
(24). MicroRNA-146a feedback inhibits RIG-I–dependent type I wild-type of miiuy croaker TAK1 39-UTR or its mutant-type reporter
IFN production by targeting IRAK1, IRAK2, and TRAF6 (25). plasmid were inserted into the mVenus-C1 (Invitrogen) which included the
More recently, the function of miRNAs in teleost fish have drawn sequence of enhanced GFP. In addition, to construct the precursor miRNA
vector, the pre-miR-217 sequence was PCR amplified and then cloned into
more and more attention. For instance, inducible miR-210 have pcDNA3.1 vector (Invitrogen). The luciferase reporter plasmid transcrip-
been reported to modulate virus-triggered immune responses tion start sites (TSS) TSS-2498, TSS-2097, TSS-1270, TSS-634, and TSS-
through NF-kB, IRF3, and JAK/STAT signaling pathways in 255, which contain the 2498-, 2097-, 1270-, 634-, and 255-bp proximal
miiuy croaker (Miichthys miiuy) (26). Miiuy croaker miR-203 promoter sequences of miiuy croaker miR-217, respectively, were con-
structed by PCR amplification using genomic DNA of miiuy croaker as
plays a crucial role in regulating the innate immune response of
templates and subsequent cloning into pGL3-basic vector (Promega).
bacterial infections by inhibiting IRAK4-mediated NF-kB sig- miR-217 promoter reporter constructs containing mutations for Ap1,
naling (27). Furthermore, miRNA-3570 has been shown to reg- Sp1, and NF-kB were constructed with specific primers. All plasmids
ulate the antibacterial and antiviral immune responses by targeting were extracted by endotoxin-free plasmid DNA Miniprep kit (Tiangen)
MyD88 and MAVS in miiuy croaker (28, 29). Collectively, these and confirmed by Sanger sequencing before using plasmids. The expression
of protein was confirmed by Western blot analysis. The sequences of all
studies have suggested the role of miRNAs as essential fine-tuning primers are listed in Supplemental Table I.
regulators involved in PRRs signaling in teleost fish. In the current
study, the function of miRNA and its relationship with TAK1- miR-217 target identification and the conservation analysis
mediated signaling in lower vertebrates have been proposed. Fur- We used TargetScan (31), miRanda (32), and MicroInspector (33) algo-
thermore, we have explored miR-217 and its relationship with rithms software-predicted miR-217 targets genes. Predictions were ranked
TAK1 following infected with Gram-negative bacteria or Siniperca based on the predicted efficacy of targeting as calculated using the context
and scores of the sites. Multiple sequence alignment of premiR-217 among
chuatsi rhabdovirus (SCRV), a typical fish RNA rhabdovirus. different species was performed using DNAMAN8.0.
Specifically, we found that miR-217 is obviously enhanced fol-
lowing Vibrio anguillarum, LPS, SCRV, and poly(I:C) treatment. mimics and inhibitors
Inducible miR-217 subsequently represses the production of in- miR-217 mimics (dsRNA oligonucleotides), miR-217 inhibitors (single-
flammatory cytokine, type I IFN, and IFN-stimulated genes (ISGs) stranded oligonucleotides chemically modified by 2’-Ome), and control
by targeting TAK1, thereby avoiding excessive inflammation. Par- oligonucleotides were commercially synthesized by GenePharma (Shanghai). All
sequence are as follows: miR-217 mimics were 59-UACUGCAUCAGGAA-
ticularly, we revealed that miR-217 could modulate the antibacterial
CUGAUUGGC-39 (sense), 59-CAAUCAGUUCCUGAUGCAGUAUU-39 (anti-
and antiviral immunity through NF-kB and IRF3 signaling path- sense); negative control mimics were 59-UUCUCCGAACGUGUCACGUTT-39
ways. To the best of our knowledge, this is the first study to dem- (sense), 59-ACGUGACACGUUCGGAGAATT-39 (antisense); miR-217 inhibi-
onstrate miR-217 as a negative feedback regulator involved in the tors were 59-GCCAAUCAGUUCCUGAUGCAGUA-39; and negative control
antibacterial and antiviral immune response in fish. inhibitors were 59-CAGUACUUUUGUGUAGUACAA-39.
RNA interference
Materials and Methods The TAK1-specific small interfering RNA (si-TAK1) sequences were 59-
Sample and challenge AGGCAAAGAUGUCGCAAUCTT-39 (sense) and 59-GAUUGCGACAU-
Miiuy croaker (∼50 g) was obtained from Zhoushan Fisheries Research CUUUGCCUTT-39 (antisense). The scrambled control RNA sequences
Institute, Zhejiang Province, China. Fish was acclimated in aerated sea- were 59-UUCUCCGAACGUGUCACGUTT-39 (sense) and 59-ACGUGA-
water tanks at 25˚C for 6 wk before experiments. The challenge experi- CACGUUCGGAGAATT-39 (antisense).
ments were performed as follows. Briefly, fish was respectively challenged Transfection and cell treatment
with 100 ml of V. anguillarum (1.5 3 108 CFU/ml), LPS (1 mg/ml;
InvivoGen), poly(I:C) (1 mg/ml; InvivoGen), or SCRV at a multiplicity of Before transient transfection, cells were seeded into 24-well or 12-well
infection (MOI) of five through i.p. As a comparison, 100 ml of physiological plates and incubated overnight. Subsequently, EPC cells were transfected
1622 miR-217 TARGETS TAK1 IN FISH

with plasmids by using X-tremeGENE HP DNA Transfection Reagent (35). To better explore the expression of miR-217 upon path-
(Roche) according to the manufacturer’s protocols. MKC cells were ogen infection, we investigated the expression of miR-217 in
transfected with RNA oligoribonucleotides by using Lipofectamine
RNAiMAX (Invitrogen) according to the manufacturer’s instructions.
miiuy croaker liver tissues following V. anguillarum or LPS
Miiuy croaker macrophages were seeded in 12-well plates overnight be- treatment. The results shown in Fig. 1A, 1B revealed that miR-
fore stimulation. Macrophages were washed and infected with LPS, 217 expression could be significantly enhanced, and reached its
poly(I:C) or SCRV at a MOI of 5, and incubated for the different time as peak at 12 or 24 h after V. anguillarum or LPS treatment, re-
indicated. spectively. Consistent with this, the expression profiles of miR-
RNA extract and quantitative real-time PCR 217 in miiuy croaker macrophages were detected and showed
significant upregulation upon LPS stimulation (Fig. 1C). Fur-
The viral RNA in the intracellular and supernatant was extracted by using
the Body Fluid Viral DNA/RNA Miniprep Kit (Axygen). Total RNA was ther validation of miR-217 expression patterns was analyzed
isolated with TRIzol Reagent (Invitrogen), and the cDNA was synthesized after RNA virus infection in vivo and in vitro. In accordance
using the FastQuant RT Kit (Tiangen), which includes DNase treatment of with the expression profiles upon Gram-negative bacterial in-
RNA to eliminate genomic contamination. The expression patterns of each fection, the levels of miR-217 were upregulated in both SCRV-
gene were performed by using SYBR Premix Ex Taq (Takara). The small
RNA was extracted by using miRcute miRNA Isolation Kit (Tiangen), and infected spleen samples and macrophages, and the highest level
miRcute miRNA FirstStrand cDNA Synthesis Kit (Tiangen) was applied to of miR-217 was expressed at 24 h postinfection in vivo and
reverse transcription of miRNAs. The expression analysis of miR-217 was in vitro (Fig. 1D, 1E). Meanwhile, poly(I:C), a synthetic analogue
executed by using the miRcute miRNA qPCR Detection Kit (Tiangen). of dsRNA, was applied as the stimulus to examine miR-217 ex-

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Real-time PCR was performed in a Applied Biosystems QuantStudio 3
pression. Similar to SCRV infection, poly(I:C) stimulation could
(Thermo Fisher Scientific). GAPDH and 5.8S rRNA were employed as
endogenous controls for mRNA and miRNA respectively as described (26). also enhance miR-217 expression in vivo and in vitro (Fig. 1F,
Primer sequences are displayed in Supplemental Table I. 1G). These data verified that miR-217 could be upregulated by
Gram-negative bacterial and SCRV infection, which indicated
Dual-luciferase report analysis
miR-217 may participate in both antibacterial and antiviral im-
To conduct miRNA target verification, the wild-type or mutant-type of mune responses.
TAK1 39-UTR luciferase reporters were cotransfected with miR-217
mimics, miR-217 inhibitors, the premiR-217 plasmid or their negative miR-217 suppresses the expression of inflammatory cytokines
controls into EPC cells. To determine the functional regulation of miR- and ISG
217, EPC cells were cotransfected with luciferase reporter genes,
TAK1 expression plasmid, pRL-TK Renilla luciferase plasmid, to- To investigate the function of miR-217 in host antibacterial and
gether with miR-217 mimics, premiR-217 plasmid, or negative con- antiviral immunity, we examined the effect of overexpression or
trols for dual-luciferase reporter assays. Afterwards, the cells were inhibition of miR-217 on the production of inflammatory cyto-
lysed for reporter activity testing using the Dual-Luciferase Reporter
kines, type I IFN, and ISGs. To this end, the effect of synthetic
Assay System (Promega). All the luciferase activity values were
achieved against the Renilla luciferase control. For each experiment, miR-217 mimics and inhibitors on miR-217 expression was first
three independent experiments were conducted, and each experiment evaluated in MKC cells. miRNA mimics are synthetic dsRNAs
was done in triplicates. that simulate naturally occurring mature miRNAs, whereas
Western blotting miRNA inhibitors are chemically modified antisense ssRNAs that
sequester and inhibit intracellular miRNAs. As expected, miR-
Cellular lysates were generated by using 13SDS-PAGE loading buffer. 217 mimics obviously enhanced miR-217 expression levels,
Proteins were extracted from cells and measured with the BCA Protein
Assay kit (Vazyme), then subjected to SDS-PAGE (10%) gel and trans- whereas miR-217 inhibitors decreased miR-217 expression levels
ferred to PVDF (Millipore) membranes by semidry blotting (Bio-Rad (Fig. 2A). Afterwards, the effects of overexpression or inhibition
Trans Blot Turbo System). The membranes were blocked with 5% BSA. of miR-217 on the expression levels of inflammatory cytokine
Protein was blotted with different Abs. The Ab against TAK1 was diluted were investigated in LPS-stimulated MKC cells. As shown in
at 1: 400 (ProteinTech), anti-Flag and b-actin mAb were diluted at 1: 2000
(Sigma-Aldrich); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG
Fig. 2B, the results showed that transfection of miR-217 mimics
(Abbkine) at 1:5000. The results were the representative of three inde- could inhibit the expression levels of LPS-induced TNF-a,
pendent experiments. The immunoreactive proteins were detected by using IL-1b, IL-6, and IL-8. On the contrary, inhibition of endog-
WesternBright ECL (Advansta). The digital imaging was performed with a enous miR-217 significantly increased the indicated inflam-
cold charge-coupled device camera.
matory cytokines expression compared with transfection of
Virus yield quantification control inhibitors.
To explore the role of miR-217 in host antiviral immunity, we
MKC cells were transfected with RNA oligonucleotides and then infected
with SCRV (MOI = 5). A volume of 0.1 ml of the cultural supernatant was then investigated the effect of miR-217 on the regulation of IFN
then serially diluted on the monolayer of EPC cells, and EPC cells were and ISGs after SCRV infection. To this end, MKC cells were
seeded into 96-well plates 24 h before measurement. The 50% tissue culture transfected with miR-217 mimics or miR-217 inhibitors, then
infectious dose (TCID50) was measured after 72 h. infected with SCRV. As shown in Fig. 2C, the results showed that
Statistical analysis miR-217 mimics decreased the expression levels of SCRV-
induced antiviral genes, including IFN-2, Mx1, ISG15, TNF-a,
Data are expressed as the mean 6 SE from at least three independent
triplicated experiments. Student t test was used to evaluate the data. The and IL-8, whereas miR-217 inhibitors upregulated the expression
relative gene expression data were acquired using the 2ΔΔCT method, and levels of the indicated genes. To further investigate the function
comparisons between groups were analyzed by one-way ANOVA followed of miR-217 in modulating RNA virus-induced antiviral immu-
by Duncan multiple comparison tests (34). A p value ,0.05 was consid- nity, poly(I:C) was used to stimulate MKC cells, then the ex-
ered significant.
pression of antiviral genes were tested. As shown in Fig. 3D,
miR-217 mimics significantly decreased, whereas miR-217 in-
Results hibitor increased the expression levels of IFN-2, Mx1, ISG15,
miR-217 is upregulated upon Gram-negative bacterial and TNF-a, and IL-8 in poly(I:C)–stimulated MKC cells. Taken to-
RNA virus infection gether, these data demonstrated that miR-217 negatively reg-
Previous studies have confirmed that miR-217 expression is ob- ulates a range of responses to either Gram-negative bacterial
viously enhanced in LPS-stimulated miiuy croaker kidney tissue infection or RNA virus infection in MKC cells, which indicating
The Journal of Immunology 1623

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FIGURE 1. The expression profiles of miR-217 following Gram-negative bacteria and RNA viral treatment. (A) The expression profiles of miR-217 in
miiuy croaker liver samples were measured by qRT-PCR at indicated time after V. anguillarum infection. (B and C) The expression profiles of miR-217 in
LPS-stimulated miiuy croaker liver samples (B) or macrophages (C). (D and E) The expression profiles of miR-217 in SCRV-treated miiuy croaker spleen
samples (D) or macrophages (E). (F and G) The expression profiles of miR-217 in miiuy croaker liver samples (F) or macrophages (G) were measured by
qRT-PCR at indicated time after poly(I:C) stimulation. The miR-217 expression levels were all measured by qRT-PCR and normalized to 5.8S rRNA.
Results are standardized to 1 in control samples. All data represented the mean 6 SE from three independent triplicated experiments. **p , 0.01 versus the
controls. qRT-PCR, quantitative RT-PCR.

the essential role of miR-217 in both antibacterial and antiviral miR-217 and that NF-kB and Sp1 transcription factor binding
immune responses. sites are required for the transcription of miR-217.
NF-kB and Sp1 sites are significant for the transcriptional TAK1 is a target gene of miR-217
activity of miR-217 promoter To explore the potential target of miR-217, miRNA target pre-
To study the transcriptional regulation of miR-217, we first ana- diction programs were used to search for potential targets of miR-
lyzed the genomic information of miR-217 from miiuy croaker 217. Through prediction analysis, TAK1 gene harbor a standard
genome and constructed a schematic diagram to examine miR-217 target sequence for miR-217 at its 39-UTR (Fig. 4A). To assess
promoter. To investigate the fully intact promoter of miR-217, we whether miR-217 directly target miiuy croaker TAK1 gene
constructed a set of luciferase reporter plasmids upstream of the through the target site in the 39-UTR, we constructed luciferase
putative TSS of miR-217. As shown in Fig. 3A, the highest lu- reporter plasmids with the putative TAK1 39-UTR target site for
ciferase activity was produced by the luciferase reporter TSS-1270 miR-217 and the mutant version that mutated of miR-217 tar-
compared with that achieved with the pGL3-basic empty plasmid, geting sequences. Luciferase reporter plasmids together with miR-
indicating TSS-1270 possessing fully intact promoter activity. 217 mimics or negative control mimics were transfected into EPC
However, transfection with TSS-634 and TSS-255 reporter plas- cells. A significant decrease in relative luciferase activity was
mids led to lower basal promoter activity, which indicates that the noted when the wild-type of TAK1-39UTR was cotransfected with
complete promoter requires the regional activity between 21270 miR-217 mimics, whereas no change of luciferase activity was
and 2634 and multiple cis-acting element promoter regions co- observed in cells transfected with the mutant-type construct
ordinately regulate promoter activity. Meanwhile, we also found (Fig. 4B). Furthermore, as shown in Fig. 4C, miR-217 mimics and
that transfection with TSS-2498 and TSS-2097 reporter plasmids inhibitors were used to further verify the downregulation mecha-
also resulted in lower basal promoter activity, which indicates the nism, and the results revealed that the inhibition of luciferase
region from 22498 to 21270 may contain negative regulatory activity was attenuated after cotransfection with miR-217 inhibi-
elements. Furthermore, the transcription factors involved in the tors. The dose-dependent effect of miR-217 mimics on the inhi-
promoter of miR-217 were analyzed with Alibaba 2.1 software in bition of luciferase activity could also be observed from 12 to 36 h
the luciferase reporter TSS-1270. Ap1, Sp1, and NF-kB binding posttransfection (Fig. 4D). In addition, miR-217 mimics have been
sites have been predicted to be located in the promoter region revealed to downregulate GFP gene expression when the wild-type
(Fig. 3B). Then, we constructed nine mutants that mutated Ap1, of TAK1 39-UTR was cloned into mVenus-C1 vector, whereas no
Sp1, or NF-kB binding site in the TSS-1270 luciferase reporter change of GFP gene expression was observed within the mutant-
plasmid to investigate the indispensable transcription factors. As type constructs (Fig. 4E).
shown in Fig. 3C, mutation binding site of Sp1 at nucleotides 21084, Given that miRNA processing system is conserved from in-
Sp1 at nucleotides 2995, Sp1 at nucleotides 2525, Sp1 at nu- vertebrates to vertebrates (27), we constructed the premiR-217
cleotides 2501, or NF-kB at nucleotides 2175 lead to signifi- expression plasmid and then transfected it into EPC cells
cant attenuation of the promoter activity. The data indicated that (Fig. 4F). After transfection of premiR-217 plasmid, we observed
Sp1 and NF-kB sites are essential for the transcription of miR- that overexpression of premiR-217 could sharply decrease lucif-
217. Taken together, these data demonstrated that the region erase activity, whereas premiR-217 showed no effect on the lu-
from 21270 to 2634 is needed for the basal promoter activity of ciferase activity of cells transfected with the mutant of TAK1
1624 miR-217 TARGETS TAK1 IN FISH

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FIGURE 2. miR-217 suppresses the expression of inflammatory cytokines and ISGs (A) MKC cells were transfected with control mimics (NC) or miR-
217 mimics (miR-217) (left panel) and control inhibitors (NC-i) or miR-217 inhibitors (miR-217-i) (right panel). At 48 h posttransfection, miR-217 ex-
pression was measured by qRT-PCR and normalized to 5.8S rRNA. (B) MKC cells were transfected with NC, miR-217, NC-i, or miR-217-i for 48 h. Then,
the cells were treated with LPS for 6 h and the expression levels of TNF-a, IL-1b, IL-6, and IL-8 were analyzed by qRT-PCR and normalized to GAPDH.
(C and D) MKC cells were transfected with NC, miR-217, NC-i, or miR-217-i for 48 h, and the cells were infected with SCRV (C) or poly(I:C) (D) for
another 24 h. The expression levels of IFN-2, MX1, ISG15, TNF-a, and IL-8 were analyzed by qRT-PCR and normalized to GAPDH. Results are
standardized to 1 in control cells. All data are presented as the means 6 SE from three independent triplicated experiments. **p , 0.01, *p , 0.05 versus
the controls. qRT-PCR, quantitative RT-PCR.

39-UTR (Fig. 4G). When the luciferase reporter plasmids were protein and mRNA levels (Fig. 5B). To measure miR-217 function
transfected with the premiR-217 plasmid and miR-217 inhibi- in the regulation of endogenous TAK1, we transfected with miR-
tors, we observed that premiR-217 plasmid markedly decreased 217 mimics or inhibitors into MKC cells to test the levels of en-
luciferase activity, whereas the inhibition effect was attenuated dogenous TAK1. As shown in Fig. 5C, transfection of miR-217
after cotransfection with miR-217 inhibitors (Fig. 4H). The mimics significantly decreased the expression of TAK1 in a dose-
concentration and time gradient experiments were likewise dependent manner. In contrast, the introduction of miR-217 in-
conducted. As shown in Fig. 4I, the premiR-217 plasmid had an hibitors markedly enhanced TAK1 expression compared with
inhibitory effect on luciferase activity in a dose-dependent control inhibitors (Fig. 5D). Furthermore, to test whether induc-
manner, and it could significantly inhibit the luciferase activity ible miR-217 suppresses TAK1 expression during LPS stimulation
from 12 to 36 h after transfection. For further validation, as and viral infection, we performed the transfection with miR-217
shown in Fig. 4J, the results revealed that premiR-217 plasmid mimics or inhibitors into MKC cells for 48 h and then treated with
could significantly inhibit GFP expression, whereas no change in LPS, SCRV, or poly(I:C). As shown in Fig. 5E, the expression
fluorescence intensity was observed in cells transfected with the of TAK1 could be significantly enhanced after LPS, SCRV or
mutant form. Together, these results adequately demonstrated poly(I:C) treatment, and overexpression miR-217 almost abol-
that a potential target binding region for miR-217 exists in the ished upregulation of TAK1 expression in MKC cells. On the
39-UTR of miiuy croaker TAK1, and miR-217 could directly contrary, the introduction of miR-217 inhibitors further enhanced
target TAK1 gene. TAK1 levels compared with control inhibitors (Fig. 5F). Above
all, these results demonstrated that miR-217 directly targets TAK1
miR-217 posttranscriptional inhibits TAK1 expression
gene, and TAK1 expression could be regulated by miR-217 during
Given that miRNAs modulate target gene expression through either LPS stimulation or RNA virus infection.
mRNA degradation or translation inhibition, we next determined
whether miR-217 participates in the regulation of TAK1 expres- miR-217 regulates NF-kB signaling as well as IRF3 signaling
sion. To this end, we constructed TAK1 expression plasmid that via TAK1
contains the full-length coding sequence region and 39-UTR of TAK1 is widely known as pivotal upstream activators of the NF-kB
miiuy croaker TAK1, and then cotransfected with miR-217 signaling pathway (14–17). There are also studies pointing that
mimics into EPC cells. As shown in Fig. 5A, overexpression of TAK1 could induce TBK1-IRF3 activation and the expression of
miR-217 exerted a significant inhibitory effect on the expression several IFN-inducible genes (18, 19). Given miR-217 modulates
levels of TAK1 in a dose-dependent manner. Consistent with miR- the expression of inflammatory cytokines, IFN, and ISGs, we thus
217, premiR-217 also reduced the expression of TAK1 at both explored whether miR-217 affects NF-kB or IRF3 signaling. To
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FIGURE 3. NF-kB and Sp1 sites are critical for the transcription of the miR-217 promoter. (A) EPC cells were transfected with miR-217 promoter
reporter mutants containing various lengths of the miR-217 promoter region. At 24 h posttransfection, luciferase activity was measured, and the luciferase
activity of transfection of pGL3-basic vector was regarded as a control. (B) Schematic representation of the transcription binding sites. (C) EPC cells were
transfected with the wild-type miR-217 promoter reporters (TSS-1270 WT) or the mutated miR-217 promoter reporters (TSS-1270 MT) for 24 h. All the
luciferase activity was normalized to Renilla luciferase activity. All data are presented as the means 6 SE from at least three independent triplicated
experiments. **p , 0.01, *p , 0.05 versus the controls.

this end, we first examined the function of miiuy croaker TAK1 in significantly decreased the activation of the indicated luciferase
regulating NF-kB or IRF3 signaling. We transfected EPC cells reporter genes at 48 h posttransfection. Moreover, we tried to
with miiuy croaker TAK1 expression plasmid in combination with determine whether miiuy croaker premiR-217 regulates the ac-
the NF-kB, IL-1b, IL-8, IFN-2, IRF3, and ISRE reporter genes for tivity of the indicated luciferase reporter genes. We investigated
48 h. As shown by the results of dual-luciferase reporter assays, that the downregulation produced by premiR-217 was presented a
miiuy croaker TAK1 gene was sufficient to activate the NF-kB, dose-dependent manner (Fig. 7E). Taken together, these results
IL-1b, IL-8, IFN-2, IRF3, and ISRE reporter genes (Fig. 6A). For indicated that miiuy croaker TAK1 could enhance both NF-kB
further validation, the results indicated that TAK1 upregulated the and IRF3 signaling, and miR-217 negatively regulates TAK1-
indicated reported genes in a dose-dependent manner, which in- mediated NF-kB and IRF3 signaling in a manner dependent on
dicating the function of miiuy croaker TAK1 in NF-kB and IRF3 suppressing TAK1.
signaling (Fig. 6B).
Knockdown of TAK1 inhibits antibacterial and antiviral
Given that miR-217 targets TAK1 and regulates its expression,
immune responses
we then tested whether miR-217 regulates the activity of the in-
dicated luciferase reporter genes. To this end, we transfected with To confirm the contribution of TAK1 to both the antibacterial and
TAK1 expression plasmid, together with miR-217 mimics or antiviral immune response, we silenced TAK1 and examined the
negative control mimics into EPC cells. As shown in Fig. 6C, 6D, expression levels of inflammatory cytokines, IFN, and ISGs upon
miR-217 mimics markedly suppressed the activation of NF-kB, LPS, SCRV, or poly(I:C) treatment. Silenced TAK1 suppresses the
IL-1b, IL-8, IFN-2, IRF3, and ISRE induced by overexpression expression levels of endogenous TAK1 in MKC cells (Fig. 7A). As
of TAK1 compared with negative control mimics, and miR-217 shown in Fig. 7B, si-TAK1 effectively inhibited the expression
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FIGURE 4. miR-217 targets miiuy croaker TAK1 gene. (A) Sequence alignment of miR-217 and its binding sites in the 39-UTR of TAK1. (B) EPC cells
were transfected the wild-type of TAK1 39-UTR (TAK1-39UTR-WT) or the mutant-type of TAK1 39-UTR (TAK1-39UTR-MT), together with miR-217 or
NC. The luciferase activity were measured by using the dual-luciferase reporter assay system. (C) EPC cells were cotransfected with TAK1-39UTR-WT,
together with NC, miR-217, NC-i or miR-217-i for 36 h, and the luciferase activity was determined. For each transfection, the total amount of oligo-
nucleotides was controlled and normalized. (D) The concentrate gradient and time gradient experiments were conducted for miR-217 transfection. (E) EPC
cells were cotransfected with the wild or mutant-type of mVenus-TAK1-39UTR, together with NC or miR-217. At 48 h posttransfection, the fluorescence
intensity was evaluated by enzyme-labeled instrument. Original magnification 310. (F) The premiR-217 sequence and the process for its plasmid con-
struction. (G) EPC cells were transfected the wild-type of TAK1-39UTR-WT or TAK1-39UTR-MT, together with premiR-217 or pcDNA3.1 for 36 h. The
luciferase activity was measured by using the dual-luciferase reporter assay system. (H) EPC cells were cotransfected with TAK1-39UTR-WT, together with
pcDNA3.1, premiR-217, NC-i or miR-217-i for 36 h, and the luciferase activity was determined. (I) The concentrate gradient and time gradient experiments
were conducted for premiR-217 transfection. (J) EPC cells were cotransfected with the wild- or mutant-type of mVenus-TAK1-39UTR, together with
pcDNA3.1 or premiR-217. At 48 h posttransfection, the fluorescence intensity was evaluated by enzyme-labeled instrument. Original magnification 310.
All the luciferase activity was normalized to Renilla luciferase activity. All data are presented as the means 6 SE from at least three independent triplicated
experiments. **p , 0.01 versus the controls.

of TAK1 in MKC cells treated with LPS, SCRV, or poly(I:C). trends were also detected in MKC cells upon SCRV or poly(I:C)
Afterwards, MKC cells were transfected with si-TAK1 and then treatment. As shown in Fig. 7D, 7E, silenced TAK1 in MKC
stimulated with LPS. As shown in Fig. 7C, knockdown of TAK1 cells obviously reduced the expression levels of TNF-a, IFN-2,
significantly decreased the expression levels of TNF-a and IL-8 MX1, and ISG15. These results confirmed that miiuy croaker
in MKC cells stimulated with LPS. Similar downregulation TAK1 is involved in regulating antibacterial and antiviral immunity,
The Journal of Immunology 1627

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FIGURE 5. miR-217 inhibits TAK1 expression. (A and B) EPC cells were cotransfected with TAK1 expression plasmid, along with NC or miR-217 (A)
and pcDNA3.1 or premiR-217 (B). After 48 h posttransfection, the levels of TAK1 were determined by Western blotting (upper panel) or qRT-PCR (lower
panel), respectively. (C and D) MKC cells were transfected with NC or miR-217 (C) and NC-i or miR-217-i (D). After 48 h posttransfection, the levels of
TAK1 were determined by Western blotting (upper panel) or qRT-PCR (lower panel), respectively. (E and F) After transfection of NC or miR-217 (E) and
NC-i or miR-217-i (F) for 48 h, MKC cells were treated with LPS, SCRV, or poly(I:C), respectively. The mRNA levels of TAK1 was analyzed by qRT-PCR
and normalized to GAPDH. The results are standardized to 1 in control cells. All data are presented as the means 6 SE from three independent triplicated
experiments. **p , 0.01, *p , 0.05 versus the controls. qRT-PCR, quantitative RT-PCR.

and silenced TAK1 has a similar effect to that of miR-217 findings that miR-217 targets TAK1 gene also exists in other
overexpression. teleost fish, which verify that the functions of miR-217 are
conserved to some extent. In total, these results indicated that
miR-217 feedback promotes virus replication
miR-217 expression could be enhanced by both Gram-negative
To investigate the biological significance of miR-217 upregulation bacterial or RNA viral infection. Upregulated miR-217 inhibits
in SCRV-induced host cells, we examined the effect of miR-217 on inflammatory cytokine, type I IFN, and ISGs production by
SCRV replication in EPC cells. By measuring the SCRV TCID50 targeting TAK1 and subsequently suppressing NF-kB and IRF3
levels in the supernatant from the infected MKC cells, we found signaling, thereby avoiding excessive inflammation and immune
that overexpression of miR-217 increased SCRV replication, whereas responses (Fig. 10).
inhibition of miR-217 decreased SCRV replication (Fig. 8A). Con-
sistent with this, overexpression of miR-217 promoted SCRV repli-
cation, whereas inhibition of miR-217 weakened SCRV replication Discussion
in intracellular and supernatant from the infected MKC cells Fishes have been regarded to be an excellent biological model in
(Fig. 8B, 8C). These data indicated that host miR-217 is able to immunology studies as it is a representative population of lower
enhance SCRV replication. vertebrates serving as an important link to early vertebrate evo-
lution. In teleost fish, the innate immune response is a fundamental
miR-217 regulation of TAK1 is widely found in teleost fish defense mechanism and is of vital importance for the disease re-
To address the generality of our findings, we first examined the sistance (36). Teleost fish use PRRs to detect conserved PAMPs on
sequence alignment of premiR-217 from various species. Impor- arrange of microbes. Similar to the PRR signaling in mammals,
tantly, as shown in Fig. 9A, mature miR-217 displayed a high PRRs in teleost elicit innate immunity by initiating multiple in-
conservation from fish to mammals. To determine whether miR- tracellular signaling cascades and subsequent signal the tran-
217 targeting TAK1 gene exists in other fish species, we explored scription of NF-kB and IRF3/7, resulting in the production of
the results in other teleost fish, including S. ocellatus, L. crocea, inflammatory cytokines, IFNs, and a family of ISGs, which exert a
and D. rerio. To this end, luciferase reporter constructs were clearance effect on pathogen invasion (37, 38).
generated by cloning TAK1 39-UTR of S. ocellatus into pmir- Because of the significance of innate immunity in teleost fish, the
GLO vector within the mutation at the miR-217 binding site as related modulators and regulation mechanisms are particularly
a control (Fig. 9B). As shown in the dual-luciferase reporter as- important. From the first report of miRNAs in zebrafish (39), the
says, miR-217 mimics were sufficient to decrease the luciferase role of miRNAs as fine-tuning regulators of different biological
activity when cotransfected with the S. ocellatus TAK1 39-UTR processes have been gradually clarified in teleost fish. Some
reporter plasmid, whereas miR-217 mimics showed no effect on miRNAs have been proposed as key switches for activating or
the luciferase activity of cells transfected with a mutant-type inhibiting of the innate immune response. For example, miR-30e
(Fig. 9C, left panel). We also found premiR-217 plasmid pre- has been demonstrated to regulate immune responses related to
sented a dose-dependent effect on the inhibition of luciferase NF-kB activation through inhibition of IkB in EPC cells (40).
activities (Fig. 9C, right panel). Meanwhile, we found that miR- More recently, researchers also found that miR-155 could suppress
217 presented a similar effect on the inhibition of luciferase ac- the expression levels of immune-related cytokines in CIK cells
tivities when cotransfected with the L. crocea TAK1 39-UTR or and zebrafish, and mIg has been identified a novel target gene of
D. rerio TAK1 39-UTR (Fig. 9D–G). These data showed that the miR-155 in fish (41). However, these miRNAs have only been
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FIGURE 6. miR-217 regulates NF-kB and IRF3 signaling via TAK1. (A) EPC cells were cotransfected with TAK1 expression plasmid, together with
pRL-TK Renilla luciferase plasmid, and NF-kB, IL-1b, IL-8, IFN-2, IRF3, or ISRE luciferase reporter genes for 48 h, then the luciferase activity was
measured. (B) Different dose of TAK1 expression plasmid were cotransfection with the indicated luciferase reporter genes for 48 h, then the luciferase
activity was measured. (C) TAK1 expression plasmid were transfected with NC or miR-217, together with TAK1 expression plasmid and the indicated
luciferase reporter genes. Afterward, the luciferase activity was measured. (D) The time gradient experiments was conducted for transfection of miR-217.
(E) TAK1 expression plasmid were transfected with pcDNA3.1 or premiR-217, together with TAK1 expression plasmid and the indicated luciferase reporter
genes for 48 h, then the luciferase activity was measured. All the luciferase activity was normalized to Renilla luciferase activity. All data are presented as
the means 6 SE from three independent triplicated experiments. **p , 0.01, *p , 0.05 versus the controls.

identified in few teleost species, indicating that the characteriza- RNA rhabdovirus. Inducible miR-217 showed a negative effect on
tion of miRNAs in fish requires much work. Based on the strong the production of inflammatory cytokines and ISGs. We further
conservation of miRNA among all vertebrates, studies of teleost demonstrated that miR-217 targets TAK1, through which miR-217
miRNA cannot only benefit for fish, but also provide a much-needed inhibited TAK1-mediated NF-kB and IRF3 signaling. Finally, the
insight into the mammal gene regulatory networks through orthologous overexpression of miR-217 could facilitate SCRV replication. These
gene functional studies. In this study, the underlying miRNA-mediated findings suggest that miR-217 plays critical roles in both antibac-
mechanisms for regulating TAK1-mediated antibacterial and an- terial and antiviral immunity, which rich the regulatory networks of
tiviral immune responses were addressed in teleost fish. We found miRNA-mediated immune responses against pathogen infection in
that miR-217 plays as a negative regulator involved in antibacte- fish species.
rial and antiviral immunity in miiuy croaker. The expression of NF-kB is a family of transcription factors that are implicated in
miR-217 could be enhanced by Gram-negative bacteria, as well as regulating cell activation, cell proliferation, inflammation, and the
The Journal of Immunology 1629

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FIGURE 7. Knockdown of TAK1 attenuates both antibacterial and antiviral immunity. (A) MIC cells were transfected with control small interfering RNA
(siRNA) (si-NC) or si-TAK1 for 48 h. Afterwards, TAK1 protein or mRNA levels were determined by Western blotting (left panel) or qRT-PCR (right
panel). (B) MKC cells were transfected with si-NC or si-TAK1 for 48 h, then treated with LPS (left panel), SCRV, or poly(I:C) (right panel), respectively.
The expression levels of TAK1 was measured by qRT-PCR. (C) MKC cells were transfected with si-NC or si-TAK1 for 48 h, and treated with LPS for 6 h.
The expression levels of TNF-a and IL-8 were determined by qRT-PCR. (D and E) After transfection of si-NC or si-TAK1, MKC cells were treated with
SCRV or poly(I:C) for another 24 h. The expression levels of TNF-a (D), IFN-2, MX1, and ISG15 (E) were measured by qRT-PCR. All data are presented as
the means 6 SE from three independent triplicated experiments. **p , 0.01, *p , 0.05 versus the controls. qRT-PCR, quantitative RT-PCR.

immune response (42). Mammalian NF-kB family include RelA the Ser536 of p65 and thereby upregulates the transactivation activity
(p65), NF-kB–1 (p105/p50), NF-kB–2 (p100/p52), RelB, and c- of NF-kB (43, 45).
Rel, which bind as a homodimer or heterodimer within the het- As pivotal upstream activators of the NF-kB pathway, TAK1 has
erodimeric of p65/p50 complex appearing to be the most abundant been reported to be regulated by a variety of molecules. For ex-
form (43, 44). In most cases, NF-kB is mainly trapped in the ample, USP4 in HeLa cells enhances TNF-a–induced TAK1
cytoplasm in an inactive form bound to IkBa proteins, the NF-kB polyubiquitination and downregulate TNF-a–induced NF-kB ac-
inhibitors. In response to stimuli, such as TNF-a or IL-1b, TAK1 tivation (20). Recently, several studies reported the involvement of
and its adaptors TAB1/2 are recruited to the receptor proximal miRNAs in TAK1-mediated signaling. In hepatocellular carci-
signaling complex, resulting in the activation of IKK. Activated noma cells, miR-26b could suppress the TNF-a–induced NF-kB
IKK phosphorylates IkBa proteins can trigger ubiquitination and signaling through targeting TAK1 and TAB3, therefore enhanc-
degradation of IkBa, thereby releasing the p50/p65 heterodimer, ing the chemosensitivity of HCC cells (46). miR-146a, serving
translocating to the nucleus, and acting as a sequence-specific as a tumor suppressor, negatively regulates the NF-kB signal-
DNA-binding transcription factor. Meanwhile, IKK phosphorylates ing pathway via targeting TAK1, thereby significantly promoting

FIGURE 8. miR-217 regulates SCRV replication. (A) MKC cells were transfected with NC, miR-217 mimics, NC-i or miR-217-i, and infected with
SCRV at MOI 5 for 1 h and washed, then added with fresh medium. After 72 h, SCRV TCID50 in cultural supernatants was measured with EPC cells. The
qRT-PCR analysis was conducted for intracellular (B) or supernatant (C) SCRV RNA. All data are presented as the means 6 SE from at least three in-
dependent triplicated experiments. **p , 0.01, *p , 0.05 versus the controls. qRT-PCR, quantitative RT-PCR.
1630 miR-217 TARGETS TAK1 IN FISH

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FIGURE 9. miR-217-regulating TAK1 gene has been found in other teleost. (A) Sequence alignment of premiR-217 from various species in which it is
present. miR-217 sequences are shown in boxes. (B) Schematic diagram of the predicted target sites of miR-217 in 39-UTR of S. ocellatus TAK1. (C) EPC
cells were transfected with NC or miR-217, along with the wild-type or the mutant-type of S. ocellatus TAK1 39-UTR and the luciferase activity was
determined (left panel). The time gradient experiments was conducted for premiR-217 transfection (right panel). (D) Schematic diagram of the predicted
target sites of miR-217 in 39-UTR of L. crocea TAK1. (E) EPC cells were transfected with NC or miR-217, along with the wild-type or the mutant-type of
L. crocea TAK1 39-UTR and the luciferase activity was determined (left panel). The time gradient experiments was conducted for premiR-217 transfection
(right panel). (F) Schematic diagram of the predicted target sites of miR-217 in 39-UTR of D. rerio TAK1. (G) EPC cells were transfected with NC or miR-
217, along with the wild-type or the mutant-type of D. rerio TAK1 39-UTR and the luciferase activity was determined (left panel). The time gradient
experiments was conducted for premiR-217 transfection (right panel). All the luciferase activity was normalized to Renilla luciferase activity. All data are
presented as the means 6 SE from at least three independent triplicated experiments. **p , 0.01, *p , 0.05 versus the controls.

gastric cancer cell apoptosis (47). Further investigations have feedback regulator involved in host antibacterial and antiviral
reported that miR-143 could attenuate pancreatic ductal adeno- immune responses.
carcinoma progression by repressing of TAK1-mdeiated NF-kB MicroRNA-217 has been found to be dysregulated in various
and MAPK pathway (48). In this study, we found that TAK1 tumors, such as breast cancer, hepatocellular carcinoma, and
enhances inflammatory cytokine production via modulating NF- ovarian cancer. miR-217 was reported to function as an oncogene in
kB signaling in miiuy croaker. Previous studies have shown the aggressive human B cell lymphomas by targeting cell fate deter-
function of mammalian TAK1 in TBK1-IRF3/7 signaling pathway mination factor (DACH1) in breast cancer cells (49), although it
(18, 19). In this study, we demonstrated for the first time, to our can act as a tumor suppressor in hepatocellular carcinoma through
knowledge, that overexpression of fish TAK1 could induce the targeting EZF3, consequentially inhibiting tumor invasion (50). In
activation of IRF3 signaling and several IFN-inducible genes, such epithelial ovarian cancer, studies have indicated the regulation role
as MX1 and ISG15, which indicates the essential role of TAK1 in of miR-217 in both of human epithelial ovarian cancer cell lines
host antiviral immunity. Furthermore, miR-217 has been revealed and tissues, in which miR-217 plays as a tumor suppressor by
as a critical regulator in the regulation of NF-kB and IRF3 sig- targeting on insulin-like factor 1 receptor (IGF1R) (51). More
naling by targeting TAK1, which indicate miR-217 as a negative recently, miR-217 has been shown to inhibit M2-like macrophage
The Journal of Immunology 1631

7. Deng, L., C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter,

C. Pickart, and Z. J. Chen. 2000. Activation of the IkappaB kinase complex by
TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique
polyubiquitin chain. Cell 103: 351–361.
8. Skaug, B., X. Jiang, and Z. J. Chen. 2009. The role of ubiquitin in NF-kappaB
regulatory pathways. Annu. Rev. Biochem. 78: 769–796.
9. Wang, C., L. Deng, M. Hong, G. R. Akkaraju, J. Inoue, and Z. J. Chen. 2001.
TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412: 346–351.
10. Seth, R. B., L. Sun, C. K. Ea, and Z. J. Chen. 2005. Identification and charac-
terization of MAVS, a mitochondrial antiviral signaling protein that activates
NF-kappaB and IRF 3. Cell 122: 669–682.
11. Xu, L. G., Y. Y. Wang, K. J. Han, L. Y. Li, Z. Zhai, and H. B. Shu. 2005. VISA is
an adapter protein required for virus-triggered IFN-beta signaling. Mol. Cell 19:
727–740.
12. Meylan, E., J. Curran, K. Hofmann, D. Moradpour, M. Binder,
R. Bartenschlager, and J. Tschopp. 2005. Cardif is an adaptor protein in the RIG-
I antiviral pathway and is targeted by hepatitis C virus. Nature 437: 1167–1172.
13. Schenten, D., and R. Medzhitov. 2011. The control of adaptive immune re-
sponses by the innate immune system. Adv. Immunol. 109: 87–124.
14. Sato, S., H. Sanjo, K. Takeda, J. Ninomiya-Tsuji, M. Yamamoto, T. Kawai,
K. Matsumoto, O. Takeuchi, and S. Akira. 2005. Essential function for the kinase
TAK1 in innate and adaptive immune responses. Nat. Immunol. 6: 1087–1095.
15. Takaesu, G., R. M. Surabhi, K. J. Park, J. Ninomiya-Tsuji, K. Matsumoto, and

Downloaded from http://journals.aai.org/jimmunol/article-pdf/205/6/1620/1459609/ji2000341.pdf by guest on 17 June 2024

R. B. Gaynor. 2003. TAK1 is critical for IkappaB kinase-mediated activation of
the NF-kappaB pathway. J. Mol. Biol. 326: 105–115.
16. Irie, T., T. Muta, and K. Takeshige. 2000. TAK1 mediates an activation signal
from toll-like receptor(s) to nuclear factor-kappaB in lipopolysaccharide-
stimulated macrophages. FEBS Lett. 467: 160–164.
FIGURE 10. The proposed model for the mechanism by which miR-217
17. Wan, J., L. Sun, J. W. Mendoza, Y. L. Chui, D. P. Huang, Z. J. Chen, N. Suzuki,
negatively regulates inflammatory cytokine, type I IFN, and ISGs pro- S. Suzuki, W. C. Yeh, S. Akira, et al. 2004. Elucidation of the c-Jun N-terminal
duction by targeting TAK1 and suppressing NF-kB and IRF3 signaling, kinase pathway mediated by Estein-Barr virus-encoded latent membrane protein
thereby avoiding excessive inflammation and immune responses. 1. Mol. Cell. Biol. 24: 192–199.
18. Suzuki, S., Y. Zhou, A. Refaat, I. Takasaki, K. Koizumi, S. Yamaoka, Y. Tabuchi,
I. Saiki, and H. Sakurai. 2010. Human T cell lymphotropic virus 1 manipulates
polarization by suppressing secretion of IL-6 in ovarian cancer interferon regulatory signals by controlling the TAK1-IRF3 and IRF4 pathways.
J. Biol. Chem. 285: 4441–4446.
(52). In another study of pancreatic ductal adenocarcinoma, miR- 19. Sakurai, H. 2012. Targeting of TAK1 in inflammatory disorders and cancer.
217 is able to inhibit the functions of KRAS and loss of which Trends Pharmacol. Sci. 33: 522–530.
enhanced tumor proliferation (53). Additionally, miR-217 has 20. Fan, Y. H., Y. Yu, R. F. Mao, X. J. Tan, G. F. Xu, H. Zhang, X. B. Lu, S. B. Fu,
and J. Yang. 2011. USP4 targets TAK1 to downregulate TNFa-induced NF-kB
been shown to be obviously enhanced in human atherosclerotic activation. Cell Death Differ. 18: 1547–1560.
lesions, which modulates endothelial cell senescence via silent 21. Xia, Z., G. Xu, X. Yang, N. Peng, Q. Zuo, S. Zhu, H. Hao, S. Liu, and Y. Zhu.
information regulator 1 (SirT1) (54). Although miR-217 was pre- 2017. Inducible tap1 negatively regulates the antiviral innate immune response
by targeting the tak1 complex. J. Immunol. 198: 3690–3704.
viously identified to play regulatory roles in multiply cancer types, 22. Carthew, R. W., and E. J. Sontheimer. 2009. Origins and Mechanisms of miR-
few studies revealed its impacts on innate immunity upon bacterial NAs and siRNAs. Cell 136: 642–655.
23. Selbach, M., B. Schwanhäusser, N. Thierfelder, Z. Fang, R. Khanin, and
or viral infection. In the current study, miR-217 has been revealed to N. Rajewsky. 2008. Widespread changes in protein synthesis induced by
play as key switches for inhibiting of host antibacterial and antiviral microRNAs. Nature 455: 58–63.
immune responses, which indicating its essential role in avoiding 24. Jurkin, J., Y. M. Schichl, R. Koeffel, T. Bauer, S. Richter, S. Konradi,
B. Gesslbauer, and H. Strobl. 2010. miR-146a is differentially expressed by
excessive inflammation and immune responses. myeloid dendritic cell subsets and desensitizes cells to TLR2-dependent acti-
In summary, this study provides direct evidence for the function vation. J. Immunol. 184: 4955–4965.
of miR-217 in both antibacterial and antiviral responses, and miR- 25. Hou, J., P. Wang, L. Lin, X. Liu, F. Ma, H. An, Z. Wang, and X. Cao. 2009.
MicroRNA-146a feedback inhibits RIG-I-dependent Type I IFN production in
217 acts as a negative regulator in modulating the production of macrophages by targeting TRAF6, IRAK1, and IRAK2. J. Immunol. 183: 2150–
inflammatory cytokines and ISGs. Additionally, we found that 2158.
26. Xu, T., Q. Chu, J. Cui, and D. K. Bi. 2018. Rhabdovirus-inducible microRNA-
TAK1 is a novel target for miR-217, and miR-217 participates in 210 modulates antiviral innate immune response via targeting STING/MITA in
regulating TAK1-mediated NF-kB and IRF3 signaling. These re- fish. J. Immunol. 201: 982–994.
sults were also confirmed in other teleost fish, indicating that miR- 27. Xu, T., Q. Chu, J. Cui, and X. Zhao. 2018. The inducible microRNA-203 in fish
represses the inflammatory responses to Gram-negative bacteria by targeting
217 targeting TAK1 is evolutionarily conserved in fish species. IL-1 receptor-associated kinase 4. J. Biol. Chem. 293: 1386–1396.
Collectively, our findings not only enhance the understanding of 28. Chu, Q., Y. Sun, J. Cui, and T. Xu. 2017. Microrna-3570 modulates the NF-kB
miR-217 function but also provide insights into the intricate net- pathway in teleost fish by targeting MyD88. J. Immunol. 198: 3274–3282.
29. Xu, T., Q. Chu, J. Cui, and D. Bi. 2018. Inducible microRNA-3570 feedback
works host–virus interaction. inhibits the RIG-I-dependent innate immune response to rhabdovirus in teleost
fish by targeting MAVS/IPS-1. J. Virol. 92: e01594-17.
Disclosures 30. Chu, Q., and T. Xu. 2016. miR-192 targeting IL-1RI regulates the immune re-
sponse in miiuy croaker after pathogen infection in vitro and in vivo. Fish
The authors have no financial conflicts of interest. Shellfish Immunol. 54: 537–543.
31. Lewis, B. P., I. H. Shih, M. W. Jones-Rhoades, D. P. Bartel, and C. B. Burge.
2003. Prediction of mammalian microRNA targets. Cell 115: 787–798.
References 32. John, B., A. J. Enright, A. Aravin, T. Tuschl, C. Sander, and D. S. Marks. 2004.
1. Kumar, H., T. Kawai, and S. Akira. 2011. Pathogen recognition by the innate Human MicroRNA targets. [Published erratum appears in 2005 PLoS Biol.
immune system. Int. Rev. Immunol. 30: 16–34. 3:e264.] PLoS Biol. 2: e363.
2. Kawai, T., and S. Akira. 2010. The role of pattern-recognition receptors in innate 33. Rusinov, V., V. Baev, I. N. Minkov, and M. Tabler. 2005. MicroInspector: a web
immunity: update on Toll-like receptors. Nat. Immunol. 11: 373–384. tool for detection of miRNA binding sites in an RNA sequence. Nucleic Acids
3. Hyun, J., S. Kanagavelu, and M. Fukata. 2013. A unique host defense pathway: Res. 33: W696–W700.
TRIF mediates both antiviral and antibacterial immune responses. Microbes 34. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression
Infect. 15: 1–10. data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
4. Brennan, K., and A. G. Bowie. 2010. Activation of host pattern recognition Methods 25: 402–408.
receptors by viruses. Curr. Opin. Microbiol. 13: 503–507. 35. Chu, Q., Y. Sun, D. Bi, J. Cui, and T. Xu. 2017. Up-regulated of miR-8159-5p
5. Kawai, T., and S. Akira. 2011. Toll-like receptors and their crosstalk with other and miR-217-5p by LPS stimulation negatively co-regulate TLR1 in miiuy
innate receptors in infection and immunity. Immunity 34: 637–650. croaker. Dev. Comp. Immunol. 67: 117–125.
6. Seth, R. B., L. Sun, and Z. J. Chen. 2006. Antiviral innate immunity pathways. 36. Magnadóttir, B. 2006. Innate immunity of fish (overview). Fish Shellfish
Cell Res. 16: 141–147. Immunol. 20: 137–151.
1632 miR-217 TARGETS TAK1 IN FISH

37. Zou, P. F., M. X. Chang, Y. Li, N. N. Xue, J. H. Li, S. N. Chen, and P. Nie. 2016. 47. Chen, Y., B. Zhou, L. Xu, H. Fan, J. Xie, and D. Wang. 2017. MicroRNA-146a
NOD2 in zebrafish functions in antibacterial and also antiviral responses via NF- promotes gastric cancer cell apoptosis by targeting transforming growth factor
kB, and also MDA5, RIG-I and MAVS. Fish Shellfish Immunol. 55: 173–185. b-activated kinase 1. Mol. Med. Rep. 16: 755–763.
38. Collet, B., and C. J. Secombes. 2002. Type I-interferon signalling in fish. Fish 48. Huang, F. T., J. F. Peng, W. J. Cheng, Y. Y. Zhuang, L. Y. Wang, C. Q. Li,
Shellfish Immunol. 12: 389–397. J. Tang, W. Y. Chen, Y. H. Li, and S. N. Zhang. 2017. MiR-143 targeting TAK1
39. Lagos-Quintana, M., R. Rauhut, W. Lendeckel, and T. Tuschl. 2001. Identifi- attenuates pancreatic ductal adenocarcinoma progression via MAPK and NF-kB
cation of novel genes coding for small expressed RNAs. Science 294: 853–858. pathway in vitro. Dig. Dis. Sci. 62: 944–957.
40. Kwak, J. S., M. S. Kim, and K. H. Kim. 2019. Generation of microRNA-30e- 49. Chen, C. Z. 2005. MicroRNAs as oncogenes and tumor suppressors. N. Engl. J.
producing recombinant viral hemorrhagic septicemia virus (VHSV) and its effect Med. 353: 1768–1771.
on in vitro immune responses. Fish Shellfish Immunol. 94: 381–388. 50. Su, J., Q. Wang, Y. Liu, and M. Zhong. 2014. miR-217 inhibits invasion of
41. Hua, Y., J. Zhang, Z. Jia, J. Li, X. Xiong, and Y. Xiong. 2019. Immune-related hepatocellular carcinoma cells through direct suppression of E2F3. Mol. Cell.
genes response to stimulation of miR-155 overexpression in CIK (ctenophar- Biochem. 392: 289–296.
yngodon idella kidney) cells and zebrafish. Fish Shellfish Immunol. 94: 142–148. 51. Li, J., D. Li, and W. Zhang. 2016. Tumor suppressor role of miR-217 in human
42. Baker, R. G., M. S. Hayden, and S. Ghosh. 2011. NF-kB, inflammation, and epithelial ovarian cancer by targeting IGF1R. Oncol. Rep. 35: 1671–1679.
metabolic disease. Cell Metab. 13: 11–22. 52. Jiang, B., S. J. Zhu, S. S. Xiao, and M. Xue. 2019. MiR-217 inhibits M2-like
43. Perkins, N. D. 2012. The diverse and complex roles of NF-kB subunits in cancer. macrophage polarization by suppressing secretion of interleukin-6 in ovarian
Nat. Rev. Cancer 12: 121–132. cancer. Inflammation 42: 1517–1529.
44. Xu, T., Q. Chu, J. Cui, and R. Huo. 2018. MicroRNA-216a inhibits NF-kB- 53. Zhao, W. G., S. N. Yu, Z. H. Lu, Y. H. Ma, Y. M. Gu, and J. Chen. 2010.
mediated inflammatory cytokine production in teleost fish by modulating p65. The miR-217 microRNA functions as a potential tumor suppressor in
Infect. Immun. 86: e00256-18. pancreatic ductal adenocarcinoma by targeting KRAS. Carcinogenesis 31:
45. Ben-Neriah, Y., and M. Karin. 2011. Inflammation meets cancer, with NF-kB as 1726–1733.
the matchmaker. Nat. Immunol. 12: 715–723. 54. Menghini, R., V. Casagrande, M. Cardellini, E. Martelli, A. Terrinoni, F. Amati,
46. Zhao, N., R. Wang, L. Zhou, Y. Zhu, J. Gong, and S. M. Zhuang. 2014. MicroRNA- M. Vasa-Nicotera, A. Ippoliti, G. Novelli, G. Melino, et al. 2009. MicroRNA 217

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26b suppresses the NF-kB signaling and enhances the chemosensitivity of hepa- modulates endothelial cell senescence via silent information regulator 1. Circulation
tocellular carcinoma cells by targeting TAK1 and TAB3. Mol. Cancer 13: 35. 120: 1524–1532.