MicroRNA-302 Cluster Downregulates Enterovirus 71–Induced Innate Immune Response by Targeting KPNA2

Nanfang Peng, Xuecheng Yang, Chengliang Zhu, Li Zhou, Haisheng Yu, Mengqi Li, Yong Lin, Xueyu Wang, Qian Li, Yinglong She, Jun Wang, Qian Zhao, Mengji Lu, Ying Zhu and Shi Liu
J Immunol July 1, 2018, 201 (1) 145-156; DOI: https://doi.org/10.4049/jimmunol.1701692

Abstract

Enterovirus 71 (EV71) induces significantly elevated levels of cytokines and chemokines, leading to local or systemic inflammation and severe complications. As shown in our previous study, microRNA (miR) 302c regulates influenza A virus–induced IFN expression by targeting NF-κB-inducing kinase. However, little is known about the role of the miR-302 cluster in EV71-mediated proinflammatory responses. In this study, we found that the miR-302 cluster controls EV71-induced cytokine expression. Further studies demonstrated that karyopherin α2 (KPNA2) is a direct target of the miR-302 cluster. Interestingly, we also found that EV71 infection upregulates KPNA2 expression by downregulating miR-302 cluster expression. Upon investigating the mechanisms behind this event, we found that KPNA2 intracellularly associates with JNK1/JNK2 and p38, leading to translocation of those transcription factors from the cytosol into the nucleus. In EV71-infected patients, miR-302 cluster expression was downregulated and KPNA2 expression was upregulated compared with controls, and their expression levels were closely correlated. Taken together, our work establishes a link between the miR-302/ KPNA2 axis and EV71-induced cytokine expression and represents a promising target for future antiviral therapy.

Introduction

Enterovirus 71 (EV71), a member of the genus Enterovirus of the Picornaviridae family, is a nonenveloped virus with positive and single-stranded RNA of ∼7400 nt that encodes a large polyprotein with a single open-reading frame flanked by a 5′-untranslated region (UTR) and a 3′-UTR (1). EV71 is transmitted via the oral–fecal route and is the major etiological agent of hand, foot, and mouth disease (HFMD) (2). Normally, EV71 infects infants and children <5 y old and usually results in mild and self-limiting disease characterized by herpangina, sore throat, and fever (3). However, EV71 infection may occasionally lead to severe complications, such as aseptic meningitis, brain stem encephalitis, and acute flaccid paralysis, a polio-like syndrome (4). As with many acute viral infections, EV71-infected patients carry high levels of cytokines and chemokines, leading to cell or tissue injury (5). The mechanism of inflammatory pathogenesis after EV71 infection has not been established.

Macromolecules, such as transcription factors, transport between the nucleus and the cytoplasm through a process termed nucleocytoplasmic transport, which is conserved across species (6). In this process, a cargo molecule generally possesses a nuclear localization signal (NLS) (7). Importin proteins specifically recognize the NLS to allow the cargo proteins to pass through the nuclear pore complex between the cytoplasm and the nucleus (7). Up to now, two types of importin have been discovered: importin α, which includes 7 members in mammals, and importin β, which includes >20 members in mammals (8). In the classical nuclear import pathway, an NLS in the cargo protein is first recognized by the adaptor protein importin α, which in turn is bound by importin β to form a complex that is transported through the nuclear pore complex (9). Once in the nucleus, the cargo protein is released when Ran-GTP interacts with importin β. Karyopherin α2 (KPNA2), also named importin α1, is a member of the importin α family (10). It weighs ∼58 kDa and comprises 529 aa. KPNA2 plays an important role in mediating transcription factor nucleocytoplasmic transport. It can recognize the NLS of p65, IRF1, TP53, and ERK1/2 and act as an adaptor to deliver cargo proteins to the nucleus (10).

MicroRNAs are endogenous small, noncoding RNAs that range from 19 to 25 nt in length (11). MicroRNAs regulate gene expression by targeting sequences with partial complementarity in the 3′-UTRs of mRNAs, resulting in mRNA degradation, mRNA cleavage, and translational repression (12). Increasing evidence indicates that viruses can regulate cellular microRNAs to facilitate progression of their life cycles. Our previous studies showed that microRNA (miR)-302c regulates influenza A virus (IAV)–induced IFN expression by targeting NF-κB–inducing kinase (13). In the current study, we determined that the miR-302 cluster, including miR-302a, miR-302b, miR-302c, and miR-302d, plays an important role in the EV71-induced innate immune response. miR-302 a/b/c/d potentially targets KPNA2, which also participates in EV71-mediated JNK1/JNK2/p38 nuclear transport. This study provides new insights into microRNA expression and EV71-regulated signaling pathways.

Materials and Methods

Ethics statement

The collection of human PBMCs from blood samples was conducted according to the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of the College of Life Sciences of Wuhan University in accordance with guidelines for the protection of human subjects. All study participants provided written informed consent for the collection of samples and subsequent analyses.

Cell culture and virus

HEK293T and RD cell lines were cultured in DMEM (Life Technologies, Waltham, MA), supplemented with 10% FBS (Life Technologies, Grand Island, NY), 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate. THP-1 cell lines were cultured in RPMI 1640 medium (Life Technologies) with 10% FBS (Life Technologies). THP-1 cells were seeded into cell plates and were incubated with PMA (50 nM) for 24 h. The differentiated THP-1–derived macrophages (t-MØ) attached to the plate bottom and were subsequently washed with fresh media for use. All cell lines were grown at 37°C in an aerobic incubator with 5% CO2.

In this study, we used the Xiangyang strain of EV71 (GenBank accession number JN230523.1, https://www.ncbi.nlm.nih.gov/nuccore/358424823/), which was previously isolated by our group (5). Cells were infected with EV71 at a multiplicity of infection (MOI) of five in serum-free conditions. The virus was adsorbed at 37°C for 1 h, and the unbound virus was washed away. Infected cells were cultured in fresh medium supplemented with 2% FBS. Virus titration was performed using RD cells in 96-well plates and expressed as the 50% tissue culture infectious dose per unit volume. EV71 titers were fitted to the Poisson model, in which 1 50% tissue culture infectious dose/ml = 0.69 PFU/ml, as described previously (14).

Reagents

Abs against human KPNA2 (ab84440), human β-tubulin (ab6046), human Lamin B (ab133741), human JNK1 (ab110724), human JNK2 (ab76125), human p38 (ab197348), Flag (ab49763), and Myc (ab32072) were purchased from Abcam. Abs against human c-Jun (9165), human c-Fos (2250), human p-c-Jun (8752), and human p-c-Fos (5348) were purchased from Cell Signaling Technology. Chemically synthesized microRNA mimics and microRNA inhibitors were purchased from Ambion (Austin, TX). A multiplex ELISA was performed on supernatants using ELISA kits (R&D Systems). TRIzol, Lipfectamine-2000, and Enzyme MIX were purchased from Invitrogen (Basel, Switzerland). Unless specified otherwise, all biochemical reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Quantitative real-time RT-PCR

Quantitative real-time PCR was described previously (15, 16). Briefly, total RNA was extracted using TRIzol reagent, according to the manufacturer’s protocol. cDNA was synthesized with the Prime-Script RT reagent Kit (TaKaRa). The expression of mature microRNAs was assayed using TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA). A two-step quantitative RT-PCR was employed with specific primers for microRNAs designed by Applied Biosystems. U6 small nuclear RNA was amplified as an internal control. Real-time PCR analyses for IL-6, TNF-α, CCL3, KPNA2, and β-actin were performed using SYBR Premix Ex Taq (Applied Biosystems). The following primers were used: 5′-CTTCTCGAACCCCGAGTGAC-3′ and 5′-ATGAGGTACAGGCCCTCTGA-3′ for TNF-α; 5′-TGGTGGATGTTCCCCCCGAG-3′ and 5′-TCCTGGGAATACTGGCACGG-3′ for IL-6; 5′-AGTTCTCTGCATCACTTGCTG-3′ and 5′-CGGCTTCGCTTGGTTAGGAA-3′ for CCL3; 5′-ATTGCAGGTGATGGCTCAGT-3′ and 5′-CTGCTCAACAGCATCTATCG-3′ for KPNA2; 5′-GGACTTCGAGCAAGAGATGG-3′ and 5′-AGGAAGGAAGGCTGGAAGAG-3′ for β-actin. Real-time PCR was performed using an ABI 7900 real-time PCR machine. The relative expression of each gene was calculated and normalized using the 2−ΔΔCt.

Western blot analysis

Western blot analyses were described in a previous study (17). Briefly, cells were harvested by low-speed centrifugation and washed with PBS. Cells were lysed in radio immunoprecipitation assay buffer (Cell Signaling Technology), and protein concentrations were determined by using bicinchoninic acid assays (Cell Signaling Technology). Forty micrograms of each protein sample were separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 1 × TBST and 5% (w/v) nonfat milk for 1 h at room temperature. Then, the membrane was incubated with primary Abs overnight at 4°C. Following an incubation with an HRP-linked secondary Ab (Jackson ImmunoResearch) for an additional 1 h. Immunoreactive bands were visualized using an ECL system (GE Healthcare).

Transfection and luciferase reporter gene assays

Cells were seeded on 24-well dishes and transfected by using Lipofectamine 3000 (Invitrogen) for 24 h, after which they were serum starved for an additional 24 h prior to harvest. A Renilla luciferase reporter vector pRL-TK was used as an internal control. Luciferase assays were performed with a dual-specific luciferase assay kit (Promega, Madison, WI). Firefly luciferase activities were normalized on the basis of Renilla luciferase activities.

Coimmunoprecipitation

Coimmunoprecipitation analyses were performed, as previously described (18). Briefly, after treatment, cells were collected and lysed using lysis buffer (20 mM Tris, pH 7, 0.5% [v/v] Nonidet-P40, 25 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM phenylmethyl sulfonyl fluoride, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, and 1 mg/ml leupeptin). Lysates were mixed and were precipitated with Abs or IgG mixed with protein G–agarose beads by overnight incubation at 4°C. Beads were washed three to five times with lysis buffer, and bound proteins were separated by SDS-PAGE with subsequent immunoblotting analysis.

PBMC isolation and transfection

PBMCs were isolated from venous blood of healthy volunteers. The PBMC fraction was obtained by density centrifugation of the blood diluted 1:1 in pyrogen-free saline over Histopaque (Sigma). The cells were gently collected, washed twice in PBS, and suspended in culture medium (RPMI 1640) at 37°C in 5% CO2. PBMCs were transfected with plasmid DNA by electroporation with the Amaxa Nucleofector II Device according to the manufacturer’s protocols.

Mammalian two-hybrid analysis

293T cells were transfected with the luciferase reporter plasmid pG5-luc (Promega), test plasmids, negative control plasmids, and positive control plasmids. Forty-eight hours posttransfection, cells were harvested, and luciferase activities were assayed using the luciferase reporter assay system (Promega), according to the manufacturer’s recommendations.

Immunofluorescence

The target cells were fixed with equal volumes of methyl alcohol and acetone for 15 min, washed three times with PBS, and blocked with PBS containing 4% BSA for 1 h at room temperature. Then, the cells were incubated with primary Ab at 4°C overnight and subsequently were incubated with Alexa492-labeled secondary Abs (ProteinTech Group) for 1 h. Mounting was performed with Vectashield mounting medium with DAPI (Vector Laboratories), and the cells were visualized by confocal laser microscopy (FLUOVIEW FV1000; Olympus, Tokyo, Japan).

Nuclear extraction

Cells were incubated in serum-free media for 24 h, washed twice with cold PBS, and scraped into 1 ml cold PBS. Cells were harvested by centrifugation (15 s) and incubated in two packed cell volumes of buffer A (10 mM HEPES, pH 8, 0.5% Nonidet P-40, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 200 mM sucrose) for 5 min at 4°C with flipping of the tube. The crude nuclei were collected by centrifugation (30 s); pellets were rinsed with buffer A, resuspended in one packed cell volume of buffer B (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, and 1.0 mM DTT), and incubated on a shaking platform for 30 min at 4°C. Nuclei were centrifuged (5 min), and supernatants were diluted 1:1 with buffer C (20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, and 1 mM DTT). Cocktail protease inhibitor tablets were added to each type of buffer. Nuclear extracts were snap-frozen in liquid nitrogen and stored at −70°C until use.

Statistical analysis

The data presented in this article were obtained from three independent reproducible experiments. The results are presented as the mean ± SD. Student t test was performed for statistical comparisons between two groups. One-way ANOVA was used to compare three or more groups. Kaplan–Meier analysis was used for the survival analysis. A p value <0.05 was considered significant and was indicated with an asterisk symbol (*).

Results

The miR-302 cluster regulates EV71-induced cytokine expression

Based on the results from our previous studies, IAV infection induces IFN-β mRNA expression by downregulating miR-302c expression (13). Because miR-302 cluster members (302a, 302b, 302c, and 302d) share a high-sequence homology, differing only in the 3′-hexanucleotides (Supplemental Fig. 1A), we investigated the role of miR-302 cluster members in EV71-triggered innate immune response. We first explored the efficiency of either overexpression or suppression of miR-302 cluster members by oligonucleotide mimics or inhibitors. Real-time RT-PCR results showed that transfection with the mimics of miR-302 cluster members had higher corresponding miR-302 expression compared with those transfected with negative controls (Supplemental Fig. 1B). By contrast, the inhibitor of miR-302 cluster members could lead to a reduction of corresponding miR-302 levels compared with controls (Supplemental Fig. 1C).

A previous study has found that EV71 strongly induces the production of IL-6, TNF-α, and CCL3 in t-MØ (19). We therefore investigated whether miR-302a, b, c, d, and s (miR-302s, the microRNAs mixed with miR-302a, b, c, and d mimics by same concentration) affected EV71-triggered production of those three cytokines. As shown in Fig. 1A, transfection of t-MØ with miR-302a, b, c, d, and s mimics led to a significant decrease in the production of EV71-induced IL-6, TNF-α, and CCL3. Decreased levels of those three cytokines in culture supernatants were also observed using ELISAs (Fig. 1A). Conversely, the miR-302 a, b, c, d, and s inhibitors increased the levels of EV71-induced IL-6, TNF-α, and CCL3 mRNAs and proteins (Fig. 1B). The role of miR-302 cluster on EV71-induced IL-6, TNF-α, and CCL3 was confirmed by repeating the experiments using RD cells (Supplemental Fig. 1D, 1E). In addition, the effect of EV71 infection on the expression of miR-302a, b, c, and d was also investigated. t-MØ were infected with different concentrations of EV71 for 12 h. Levels of miR-302 a, b, c, and d were detected by real-time RT-PCR, and the levels of miR-302 a, b, c, and d were decreased by EV71 infection in a dose-dependent manner (Fig. 1C). Consistently, real-time RT-PCR results showed that EV71 infection also downregulated miR-302 a, b, c, and d expression in a time-dependent manner (Fig. 1D). Subsequently, the effect of TLR ligands on the expression of miR-302 cluster was determined. The expression of miR-302 cluster was decreased by LPS (TLR4), poly(I:C) (TLR3), and R-848 (TLR7) and TLR9 (CpG ODN) (Fig. 1E–H). Moreover, there was no association between miR-302 cluster expression and IFN-α or IFN-β treatments (Fig. 1I). Oligoadenylate synthetase 2 detection was included as a positive control (Fig. 1J). Taken together, these results show that miR-302 cluster indeed plays an important role in EV71-triggered innate immune response.

FIGURE 1.
FIGURE 1.

The miR-302 cluster inhibits proinflammatory cytokine production in response to EV71 infection. (A) t-MØ were transfected with the indicated microRNA mimics or mimic controls for 24 h, infected with EV71 (MOI = 5) or mock infected for 12 h, and subjected to real-time RT-PCR (left panel) and ELISA (right panel). (B) Experiments were performed similar to those described in (A), except that the indicated microRNA inhibitor or inhibitor control was used. (C) t-MØ were infected with the indicated concentrations of EV71 for 12 h. Levels of the miR-302 cluster were quantified using real-time RT-PCR. (D) t-MØ were infected with EV71 (MOI = 5) for the indicated times. Levels of the miR-302 cluster were quantified using real-time RT-PCR. (E–H) RD cells were transfected with TLR3 (E), TLR4 (F), TLR7 (G), and TLR9 (H). Twelve hours later, transfected cells were stimulated or unstimulated (Med) for 12 h with poly(I:C) (50 μg/ml) (E), LPS (100 ng/ml) (F), R-848 (10 nM) (G), and CpG DNA (1 μM) (H) and subjected to real-time RT-PCR analyses. (I) RD cells were stimulated or unstimulated with IFN-α (500 IU/ml) (left panel) or IFN-β (500 IU/ml) (right panel) for 12 h and subjected to real-time RT-PCR analyses. (J) Experiments were performed similar to those described in (E)–(I), except that OAS2 expression was analyzed by real-time RT-PCR. In the real-time RT-PCR experiments, the control was designated 1. The expression levels were normalized to the U6 small nuclear RNA or β-actin. Bar graphs are presented as the mean ± SD, n = 3. *p < 0.05, **p < 0.01.

KPNA2 as a direct target gene of miR-302 cluster

Because miR-302 a, b, c, and d were likely involved in EV71-triggered innate immune response, we sought to elucidate the molecular mechanisms by which miR-302 a, b, c, and d execute their functions. Using computational tools (TargetScan, miRanda, and TargetRank), we predicted that the 3′-UTR of the KPNA2 mRNA contains one miR-302 cluster binding site and then constructed wild-type KPNA2 3′-UTR and mutated reporter plasmids (Fig. 2A). The results of the luciferase activity assay demonstrated that the miR-302 a, b, c, d, and s mimics reduced the activity that is controlled by the KPNA2 3′-UTR, but the attenuation of luciferase activity was impaired by KPNA2 3′-UTR–binding site mutation (Fig. 2B). Conversely, miR-302 a, b, c, d, and s inhibitors induced KPNA2 3′-UTR luciferase activity, and miR-302 a, b, c, and d binding site mutations in KPNA2 3′-UTR disrupted the induction by miR-302 a, b, c, d, and s inhibitors (Fig. 2C). We next examined the effects of miR-302 a, b, c, and d on endogenous KPNA2 expression. The results of real-time RT-PCR and a Western blot assay demonstrated that the miR-302 a, b, c, d, and s mimics significantly decreased KPNA2 mRNA and protein expression (Fig. 2D) and, conversely, that the miR-302 a, b, c, d, and s inhibitors induced KPNA2 mRNA and protein expression (Fig. 2E). Altogether, the data indicated that the miR-302 cluster downregulates KPNA2 expression by directly targeting its 3′-UTR.

FIGURE 2.
FIGURE 2.

KPNA2 is a target of the miR-302 cluster. (A) Predicted miR-302 cluster binding sites in the 3′-UTR of the KPNA2 mRNA. Perfect matches in seed regions are indicated by red-colored words. Mutations (red-colored words) were generated in the binding sites in the 3′-UTR for the reporter gene assay. (B) RD cells were transfected with the wild-type or indicated mutants of the KPNA2 3′-UTR reporter plasmid and miR-302 cluster mimics for 48 h prior to luciferase assays. (C) Experiments were performed as described in (B), except that cells were transfected with the miR-302 cluster inhibitor or inhibitor controls. (D) RD cells were transfected with the miR-302 cluster mimics or mimics controls for 24 h prior to real-time RT-PCR (upper panel) and Western blot (lower panel) analyses. (E) Experiments were performed as described in (D), except that a nonspecific inhibitor control or miR-302 cluster inhibitors were used. In the real-time RT-PCR experiments, the control was designated 1. Bar graphs are presented as the mean ± SD, n = 3. **p < 0.01. n.s., not significant.

EV71 induces cytokine expression through KPNA2

Because the miR-302 cluster plays an important role in EV71-induced cytokine expression and KPNA2 is a direct target gene of the miR-302 cluster, we next examined the role of KPNA2 in EV71-induced cytokine expression. As shown in Fig. 3A, KPNA2 overexpression promotes EV71-induced production of the IL-6, TNF-α, and CCL3 mRNAs and proteins. We designed two specific small interfering RNAs (siRNAs) for KPNA2 (siRNA-KPNA2#1 and #2) and tested their efficiency (Fig. 3B). SiRNA-KPNA2#2 was selected for the experiments described below. The results from real-time RT-PCR and ELISA assays indicated that KPNA2 knockdown inhibited EV71-induced production of the IL-6, TNF-α, and CCL3 mRNAs and proteins (Fig. 3C). EV71-induced cytokine expression through KPNA2 was not cell-type specific because similar results were observed in RD cells (Fig. 3D, 3E). In addition, the effect of EV71 infection on the expression of KPNA2 was also investigated. t-MØ were infected with different concentrations of EV71 for 12 h. Levels of KPNA2 were detected by real-time RT-PCR and Western blot, and the levels of KPNA2 were increased by EV71 infection in a dose-dependent manner (Fig. 3F). Consistently, real-time RT-PCR and Western blot results showed that EV71 infection also upregulated KPNA2 expression in a time-dependent manner (Fig. 3G). These observations strongly suggest that KPNA2 is involved in EV71-induced cytokine expression.

FIGURE 3.
FIGURE 3.

Effects of KPNA2 on EV71-induced inflammatory cytokine expression. (A and B) t-MØ were transfected with the indicated expression constructs or empty vector for 24 h, infected with EV71 (MOI = 5) or mock infected for 24 h, and subjected to real-time RT-PCR (left panel) and ELISA (right panel). (B) RD cells were transfected with the indicated KPNA2 siRNAs or siRNA control for 48 h prior to real-time RT-PCR (upper panel) and Western blot (lower panel) analyses. (C) Experiments were performed similar to those described in (A), except that KPNA2 siRNAs or siRNA control was used. (D and E) Experiments were performed as in (A) and (C), except that RD cells were used. (F) t-MØ were infected with the indicated concentrations of EV71 for 12 h. KPNA2 RNA levels were quantified by real-time RT-PCR (upper panel), and protein levels were detected by Western blotting (lower panel). (G) t-MØ were infected with EV71 (MOI = 5) for the indicated times. KPNA2 RNA levels were quantified by real-time RT-PCR (upper panel), and protein levels were detected by Western blotting (lower panel). All experiments were repeated at least three times for consistent results. Bar graphs are presented as the mean ± SD, n = 3. *p < 0.05, **p < 0.01. n.s., not significant.

The miR-302 cluster inhibits EV71-stimulated KPNA2 and cytokine expression

Because both the miR-302 cluster and KPNA2 are involved in EV71-induced cytokine expression, we next examined the role of the miR-302 cluster on EV71-induced KPNA2 expression. The results from real-time PCR and Western blot analysis showed that transfection with miR-302 a, b, c, and d mimics decreases EV71-mediated induction of KPNA2 mRNA and protein expression (Fig. 4A). In contrast, high levels of KPNA2 mRNA and protein are present in t-MØ, when the expression of miR-302 a, b, c, and d is inhibited by microRNA inhibitors (Fig. 4B). We next examined the functional relevance of the miR-302 cluster/KPNA2 interaction in EV71-induced cytokine expression. Results from real-time RT-PCR and ELISA assays confirmed that overexpression of KPNA2 rescued the inhibitory effects of the miR-302 cluster on the production of EV71-induced IL-6, TNF-α, and CCL3 (Fig. 5A, 5B). Conversely, low levels of IL-6, TNF-α, and CCL3 were observed in t-MØ cells when KPNA2 was knocked down (Fig. 5C, 5D). Thus, the miR-302 cluster is required for EV71-induced KPNA2 and cytokine expression.

FIGURE 4.
FIGURE 4.

EV71 induces KPNA2 expression via the miR-302 cluster. (A) RD cells were transfected with the miR-302 cluster mimic or mimic controls for 24 h, infected with the EV71 (MOI = 5) or mock infected for 12 h, and subjected to real-time RT-PCR (upper panel) and Western blot (lower panel) analyses. (B) Experiments were performed similar to those described in (A), except that the miR-302 cluster inhibitor or inhibitor controls were used. All experiments were repeated at least three times for consistent results. In the real-time RT-PCR experiments, the control was designated 1. Bar graphs are presented as the mean ± SD, n = 3. **p < 0.01. n.s., not significant.

FIGURE 5.
FIGURE 5.

miR-302 cluster regulates EV71-induced inflammatory cytokine expression via KPNA2. (A and B) t-MØ were transfected with the indicated expression constructs or microRNA mimic for 24 h, infected with EV71 (MOI = 5) or mock infected for 12 h, and subjected to real-time RT-PCR (A) and ELISA (B). (C and D) Experiments were performed as in (A) and (B), except that KPNA2 siRNAs or siRNA control was used. The control was designated 1. Bar graphs are presented as the mean ± SD, n = 3. **p < 0.01.

KPNA2 interacts with JNK1/JNK2 and p38

Because a previous study found that JNK1/JNK2 and p38 MAPK signaling pathways are positively regulated by secreted inflammatory cytokines during EV71 replication (20), we hypothesized that KPNA2 associates with JNK1/JNK2 and p38. To test this hypothesis, we evaluated the binding of KPNA2 protein to JNK1/JNK2, p38, or IRF9 in vivo using a GAL4/VP16-based mammalian two-hybrid system in 293T cells. The results showed that the levels of luciferase activity in the KPNA2 and JNK1/JNK2 or p38 cotransfection group were as high as in cells cotransfected with positive control plasmids (pM-p53 and pVP16-T; Fig. 6A). IRF9 was used as a negative control (Fig. 6A). To confirm these results, we next performed transient transfection and coimmunoprecipitation experiments to further analyze the interaction between KPNA2 protein to JNK1/JNK2, p38, or IRF9. As shown in Fig. 6B–D, Myc-tagged KPNA2 interacted with Flag-tagged JNK1/JNK2 or p38. Reverse coimmunoprecipitation experiments also indicated that Flag-tagged JNK1/JNK2 or p38 interacted with Myc-tagged KPNA2 (Fig. 6B–D). In contrast, another transcription factor, IRF9, did not interact with Us2 (Fig. 6E). We further performed endogenous coimmunoprecipitation experiments, and the results indicated that KPNA2 was associated with JNK1/JNK2 or p38 in RD cells or t-MØ infected with EV71 since 6 h; this association increased after stimulation with EV71 (Fig. 6F, 6G). These results suggest that during EV71 infection, KPNA2 associated with JNK1/JNK2 and p38 positively regulate inflammatory cytokine production.

FIGURE 6.
FIGURE 6.

KPNA2 interacts with JNK1, JNK2, and p38. (A) 293T cells were cotransfected with control plasmids, pG5-luc (a luciferase reporter plasmid), pVP16-KPNA2, and pM-JNK1, pM-JNK2, or pM-P38 for 48 h prior to the mammalian two-hybrid analysis. (B) 293T cells were transfected with the indicated plasmids. Coimmunoprecipitation and immunoblot analyses were performed with the indicated Abs. (C–E) Experiments were performed as described in (B), using Flag-JNK2 (C), Flag-p38 (D), or Flag-IRF9 (E). (F and G) RD cells (F) or t-MØ (G) were infected with EV71 for the indicated times or left uninfected. Immunoprecipitation and immunoblot analysis were performed with the indicated Abs. All experiments were repeated at least three times for consistent results. Bar graphs are presented as the mean ± SD, n = 3. **p < 0.01. ND, not detected; n.s., not significant.

KPNA2 mediates nuclear translocation of JNK1/JNK2, p38, and p65 during EV71 infection

In a previous study, we found that miR-302a inhibited IAV-stimulated IRF5 expression and cytokine storm induction (21). In this study, we analyzed the role of miR-302 cluster/IRF5 axis in EV71-induced cytokine expression. As shown in Supplemental Fig. 2A and 2B, the miR-302 cluster mimics decreased IRF5 mRNA and protein expression, and conversely, the miR-302 cluster inhibitors increased IRF5 mRNA and protein expression. Further study found that the miR-302 cluster also regulates EV71-induced cytokine expression in IRF5 knockdown cells (Supplemental Fig. 2C–E), suggesting that other transcription factors involved in the miR-302 cluster regulated signaling pathway.

Because KPNA2 is a member of the importin α family and associates with JNK1/JNK2 and p38, we investigated whether KPNA2 plays a role in the nuclear translocation of JNK1/JNK2 and p38 during EV71 infection. Immunofluorescence assays showed that JNK1/JNK2 and p38 displayed cytoplasmic distribution in unstimulated cells (Fig. 7A–C). Furthermore, translocation of these transcription factors from the cytoplasm into the nucleus was observed postinfection with EV71, but knockdown of KPNA2 inhibited EV71-induced translocation of these transcription factors (Fig. 7A–C). Similar results were obtained by Western blot analysis (Fig. 7D). In addition, the effect of KPNA2 on the degrees of total and phosphorylated JNK1/JNK2 and p38 were examined by Western blot. As shown in Fig. 7E, knockdown of KPNA2 inhibited EV71-induced JNK1/JNK2 and p38 phosphorylation. Considering that KPNA2 is a direct target gene of the miR-302 cluster, we determined whether the miR-302 cluster plays a role in translocation of JNK1/JNK2 and p38. Western blot analyses indicated that these transcription factors protein levels were lowered in the cytosol and elevated in the nucleus after EV71 infection (Fig. 7F). Overexpression of the miR-302 cluster inhibited the translocation of JNK1/JNK2 and p38 to the nucleus compared with control (Fig. 7F). In contrast, knockdown of the miR-302 cluster induced EV71-induced translocation of these transcription factors (Fig. 7G).

FIGURE 7.
FIGURE 7.

miR-302/KPNA2 axis is required for EV71-induced translocation of JNK1, JNK2, and p38. (A–C) RD cells were transfected with the indicated KPNA2 siRNAs or siRNA control for 24 h, infected with EV71 (MOI = 5) or mock infected for 12 h, and subjected to immunofluorescence assays (original magnification ×200). (D) RD cells were transfected with the indicated KPNA2 siRNAs or siRNA control for 24 h and infected with EV71 (MOI = 5) or mock infected for 12 h. Cytosolic and nuclear extracts were subjected to Western blot analyses. Lamin B and β-tubulin were used as markers for nuclear and cytosolic fractions, respectively. (E) RD cells were transfected with the indicated KPNA2 siRNAs or siRNA control for 24 h, infected with EV71 (MOI = 5) or mock infected for 6 h, and subjected to Western blot analyses. (F) RD cells were transfected with the miR-302 cluster mimic or mimic controls for 24 h and infected with EV71 (MOI = 5) or mock infected for 12 h. Cytosolic and nuclear extracts were subjected to Western blot analyses. (G) Experiments were performed similar to those described in (F), except that the miR-302 cluster inhibitor or inhibitor controls were used. All experiments were repeated at least three times with consistent results. ND, not detected.

Given that KPNA2 is also involved in the nuclear localization of p65 (10), we further evaluated the role of the miR-302 cluster/KPNA2/p65 axis in EV71-induced cytokine expression. The results from real-time RT-PCR and ELISA assays indicated that overexpression of p65 promotes EV71-induced production of the IL-6, TNF-α, and CCL3 mRNAs and proteins, and conversely, p65 knockdown inhibited EV71-induced cytokine expression (Fig. 8A, 8B). The effect of the miR-302 cluster/KPNA2 axis on EV71-induced translocation of p65 was explored. As shown in Fig. 8C, EV71 infection induced the translocation of p65 to the nucleus as expected, but transfection with miR-302 cluster mimics completely abolished effects of EV71 infection. In contrast, high levels of p65 are present in the nucleus, when the expression of the miR-302 cluster is inhibited by microRNA inhibitors (Fig. 8D). Similarly, overexpression of KPNA2 induced EV71-induced translocation of p65, whereas knockdown of KPNA2 could lead to a reduction of p65 nuclear localization (Fig. 8E, 8F). These findings demonstrate that KPNA2 regulated EV71-induced proinflammatory response by mediation of JNK1/JNK2, p38, and p65 nuclear translocation.

FIGURE 8.
FIGURE 8.

The relationship between p65 and miR-302 cluster/KPNA2 axis during EV71 infection. (A) t-MØ were transfected with the indicated expression constructs or empty vector for 24 h, infected with EV71 (MOI = 5) or mock infected for 24 h, and subjected to real-time RT-PCR (left panel) and ELISA (right panel). (B) Experiments were performed similar to those described in (A), except that p65 siRNAs or siRNA control was used. (C) RD cells were transfected with the miR-302 cluster mimic or mimic controls for 24 h and infected with EV71 (MOI = 5) or mock infected for 12 h. Cytosolic and nuclear extracts were subjected to Western blot analyses. Lamin B and β-tubulin were used as markers for nuclear and cytosolic fractions, respectively. (D) Experiments were performed similar to those described in (C), except that the miR-302 cluster inhibitor or inhibitor controls were used. (E and F) Experiments were performed similar to those described in (C) and (D), except that the indicated expression constructs or siRNAs were used. All experiments were repeated at least three times for consistent results. Bar graphs are presented as the mean ± SD, n = 3. *p < 0.05, **p < 0.01. n.s., not significant.

The expression of the miR-302 cluster and KPNA2 in EV71-infected HFMD patients

To validate the role of the miR-302 cluster and KPNA2 in EV71 infection, the expression of miR-302 a, b, c, d, and KPNA2 was analyzed in healthy individuals (n = 41) and EV71-infected HFMD patients (n = 41). As shown in Fig. 9A and 9B, the expression of the miR-302 cluster was lower and the expression level of KPNA2 was higher in HFMD patients than in healthy individuals. Interestingly, low levels of miR-302 a, b, c, and d expression were correlated with high levels of KPNA2 (Fig. 9C). These observations strongly suggest that alterations of miR-302 a, b, c, d, and KPNA2 expression could be involved in EV71-induced cytokine expression (Fig. 10).

FIGURE 9.
FIGURE 9.

Correlation between KPNA2 expression and suppressed miR-302 levels in EV71-infected HFMD patients. (A and B) Real-time RT-PCR assays of miR-302 cluster (A) and KPNA2 (B) expression levels in PBMCs from healthy individuals (n = 41) or EV71-infected HFMD patients (n = 41). (C) The relative KPNA2 mRNA and miR-302 cluster levels in PBMCs from EV71-infected HFMD patients (n = 41) were subjected to Pearson correlation analysis. Boxplots illustrate medians with 25 and 75% values, and error bars represent the 5th and 95th percentiles. For (A)–(C), the lowest value was designated 1. KPNA2 or miR-302 expression data are expressed as fold induction (fold change) relative to the lowest value. **p < 0.01.

FIGURE 10.
FIGURE 10.

Model of the biological effect of the miR-302/KPNA2 axis. EV71 induces KPNA2 expression via inhibiting miR-302 cluster expression. Enhanced KPNA2 expression interacts with JNK1, JNK2, and p38, resulting in the translocation of these transcription factors from the cytoplasm to the nucleus for subsequent production of inflammatory cytokines. TF, transcription factor.

Discussion

It is now widely accepted that virus replication combined with damaged tissues and the induction of inflammatory cytokines is a putative cause of pathogenesis (22). In EV71-infected patients, high levels of cytokine and chemokine secretion were observed in the serum and cerebrospinal fluid in patients with brainstem encephalitis and pulmonary edema, which demonstrated a significant correlation between cytokines and chemokines and disease severity (23, 24). Although the role of inflammatory cytokines in EV71-mediated disease severity has been described, the mechanisms behind this event have not been established. In this study, we identify a link between the miR-302/KPNA2 axis and EV71-induced cytokine expression. Our study reveals that the miR-302 cluster and KPNA2 are important mediators of EV71-induced cytokine expression, as overexpression or loss of the miR-302 cluster and KPNA2 severely changed EV71-induced cytokine expression. Further studies demonstrated that the miR-302 cluster regulates EV71-induced cytokine expression via targeting the 3′-UTR of KPNA2 and inhibiting KPNA2-mediated JNK1/JNK2/p38 nuclear transport. Understanding the molecular correlation between miR-302 cluster/KPNA2 activity and EV71-induced cytokine expression could lay the basis for the future development of new therapeutic protocols for EV71.

The human cluster miR-302–367 is one of the main groups of miRs, which are highly expressed in embryonic stem cells (25). It has eight members, including miR-302a, miR-302a*, miR-302b, miR-302b*, miR-302c, miR-302c*, miR-302d, and miR-367 (26). The first seven microRNAs constitute the miR-302 family with a highly conserved sequence, which are cotranscribed in a polycistronic way (27). Most studies found that the miR-302 cluster plays an important role in early embryonic development, somatic cell reprogramming, and tumorigenesis (28), but the function of miR-302 in virus infection is poorly understood. Our previous studies showed that miR-302c regulates IAV-induced IFN expression by targeting the 3′-UTR of NF-κB-inducing kinase (13). As shown in another of our previous studies, miR-302a, another member of the miR-302 cluster, suppresses IAV-stimulated IFN regulatory factor-5 expression and cytokine storm induction (21). In the current study, we observed for the first time, to our knowledge, that four of the mature microRNAs from the miR-302 cluster (miR-302a, -b, -c, and -d) play an important role in EV71-induced cytokine expression by targeting the 3′-UTR of KPNA2. It appears that, during viral infection, the miR-302 cluster controls cytokine expression via targeting multiple proteins in one signaling pathway. Interestingly, miR-99 family members were also reported to target many cellular genes, such as IGF-1R, Akt, mTOR, and Ago2, and several components of the TGF-β signaling pathway (29). To our knowledge, this phenomenon seems to combine multiple security strategies for the virus to control one signaling pathway, which plays a key role in virus infection. It is also possible that the virus developed different strategies to counteract host-induced signaling pathway responses in different stages of the viral life cycle. In addition, a previous study has found that miR-302 cluster members cooperatively sensitize breast cancer cells to adriamycin via suppressing P-gp and by targeting MEKK1 of the ERK pathway (30). However, in this study, we did not observe this phenomenon, emphasizing the need for future studies.

KPNA2, a member of the karyopherin α (also called importin α) protein family, is identified as a nucleocytoplasmic transporter (31). However, to our knowledge, only a few reports have described the relationship between KPNA2 and viral infection. KPNA2 can bind to Epstein-Barr nuclear Ag leader protein to promote EBV replication (32). KPNA2 can bind to the nonstructural protein 5 (NS5) of dengue virus, which leads to nuclear localization of dengue virus NS5 (33). KPNA2 is a cellular protein that is important for hydrolase sterile α motif domain and HD domain 1 nuclear import during HIV infection (34). Severe acute respiratory syndrome coronavirus ORF6 protein binds to KPNA2 and disrupts the STAT1 complex in the nucleus (35). In the current study, for the first time, to our knowledge, we observed that KPNA2 mediates EV71-induced cytokine expression by regulating nuclear translocation of JNK1/JNK2 and p38. However, our assay could not determine whether the interaction of KPNA2 and JNK1/JNK2 or p38 is the only mechanism regulating EV71-induced cytokine expression because KPNA2 also mediated the nuclear translocation of many additional transcription factors, such as p65, IRF1, TP53, and ERK1/2. When considering the next step, studies exploring those transcription factors in EV71-induced cytokine expression would advance our knowledge of the host immune response to EV71. A previous study has found that miR-26b inhibits tumor metastasis by targeting KPNA2 in human gastric cancer (36). We also investigated the effect of miR-26b on EV71-induced cytokine expression. The results showed that the levels of EV71-induced IL-6, TNF-α, and CCL3 mRNAs and proteins were not significantly affected by miR-26b mimics or miR-26b inhibitors (Supplemental Fig. 3). It appears that KPNA2 is an important multifunction protein and that different biological processes control the function of KPNA2 via diverse and uncorrelated pathways. In addition, it is noteworthy that neither miR-302 cluster mimics nor KPNA2 siRNA cannot entirely inhibit EV71-induced cytokine expression (Figs. 1, 3). Therefore, it should exist in some microRNA-independent mechanisms of EV71-induced cytokine expression or additional functionally relevant miR-302 targets. When considering the next step, studies exploring these questions would be of great help in further clarifying the mechanism of EV71-induced cytokine expression.

We propose a hypothetical model describing the role of the miR-302/KPNA2 axis in EV71-induced cytokine expression (Fig. 10). EV71 infection stimulates KPNA2 expression via inhibition of the expression of the miR-302 cluster, which can directly target the 3′-UTR of KPNA2. KPNA2 then interacts with JNK1/JNK2 and p38, leading to translocation of activated JNK1/JNK2 and p38 from the cytosol into the nucleus for subsequent production of cytokines. Although more studies are needed to understand the delicate regulatory mechanisms of EV71-induced cytokine expression, our findings provide evidence of a distinct role for the miR-302/KPNA2 axis in this process and point to potential novel clinical uses for the miR-302/KPNA2 axis in antiviral therapy.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by grants from the National Natural Science Foundation of China (31500149 to S.L., 31570870 to Y.Z., and 81301428 to L.Z.), the Fundamental Research Funds for the Central Universities (2042017kf0221 to S.L. and 2042015kf0188 to L.Z.), the 973 Program of the National Basic Research Program of China (2014CB542603 to S.L.), the Natural Science Foundation of Hubei Province Innovation Group (2017CFA022 to Y.Z.), the Deutsche Forschungsgemeinschaft (TRR60 and RTG1949 to M. Lu), and the Foundation of Technology Bureau of Wuxi, China (CSE31N1712 to J.W.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    EV71
    enterovirus 71
    HFMD
    hand, foot, and mouth disease
    IAV
    influenza A virus
    KPNA2
    karyopherin α2
    miR
    microRNA
    MOI
    multiplicity of infection
    NLS
    nuclear localization signal
    siRNA
    small interfering RNA
    t-MØ
    THP-1–derived macrophage
    UTR
    untranslated region.

  • Received December 7, 2017.
  • Accepted April 25, 2018.

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