MiR-10a-5p-Mediated Syndecan 1 Suppression Restricts Porcine Hemagglutinating Encephalomyelitis Virus Replication

Abstract

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a single-stranded RNA coronavirus that causes nervous dysfunction in the infected hosts and leads to widespread alterations in the host transcriptome by modulating specific microRNA (miRNA) levels. MiRNAs contribute to RNA virus pathogenesis by promoting antiviral immune response, enhancing viral replication, or altering miRNA-mediated host gene regulation. Thus, exploration of the virus-miRNA interactions occurring in PHEV-infected host may lead to the identification of novel mechanisms combating the virus life cycle or pathogenesis. Here, we discovered that the expression of miR-10a-5p was constitutively up-regulated by PHEV in both the N2a cells in vitro and mice brain in vivo. Treatment with miR-10a-5p mimics allowed miR-10a-5p enrichment and resulted in a significant restriction in PHEV replication, suggesting widespread negative regulation of the RNA virus infection by miR-10a-5p. The outcomes were also evidenced by miR-10a-5p inhibitor over-expression. Luciferase reporter, quantitative real-time PCR (qRT-PCR), and western blotting analysis further showed that Syndecan 1 (SDC1), a cell surface proteoglycan associated with host defense mechanisms, acts as a target gene of miR-10a-5p during PHEV infection. Naturally, siRNA-mediated knockdown of SDC1 leads to a reduction in viral replication, implying that SDC1 expression is likely a favorable condition for viral replication. Together, the findings demonstrated that the abundant miR-10a-5p leads to downstream suppression of SDC1, and it functions as an antiviral mechanism in the PHEV-induced disease, providing a potential strategy for the prevention and treatment of PHEV infection in the future work.

Keywords: Syndecan 1; antiviral mechanism; miR-10a-5p; porcine hemagglutinating encephalomyelitis virus; virus replication.

FIGURE 1

Porcine hemagglutinating encephalomyelitis virus (PHEV) infection up-regulates miR-10a-5p expression. (A) MiRNA array of PHEV-infected and uninfected mice. Hierarchical clustering shows distinguishable miRNA expression profiling among samples. (B) N2a cells were infected with PHEV at 2 × 10⁶ TCID₅₀/ml and harvested at 0, 24, 48, and 72 h post infection. The expression of miR-10a-5p was determined by qRT-PCR. U6 was chosen as a housekeeping gene to normalize miR-142a-3p expression. Another irrelative miR-130b-5p is used as a negative control. (C) Cells were treated as described in panel (B), then the total RNA was extracted, and the relative expression of PHEV mRNA was determined by qRT-PCR. Data were normalized to GAPDH. (D) N2a cells treated as described in panel (B), and the cell lysates were collected and subjected to examine the expression of PHEV protein by western blotting. Beta actin expression was verified as loading control. (E) BALB/c mice were infected with PHEV for 3 or 5 days, and then the cerebral cortexes were harvested, and miR-10a-5p level was determined by qRT-PCR. N = 4 mice per group, three independent experiments. (F) The lysis from cerebral cortexes of mice as described in panel (E) was collected, and then the relative expression of PHEV mRNA was determined by qRT-PCR. (G) The cerebral cortical lysis was detected by western blotting, and the PHEV protein was normalized to beta actin. All of data are representative of at least three independent experiments, with the error bars representing the standard deviations (SDs). *p < 0.05, **p < 0.01 vs. normal controls.

FIGURE 2

Abundant miR-10a-5p suppresses the replication of porcine hemagglutinating encephalomyelitis virus (PHEV). (A) N2a cells were transfected with different concentrations of miR-10a-5p mimic for 24 h, and then the expression of miR-10a-5p was analyzed by qRT-PCR and normalized to U6. (B) N2a cells were transfected with 100 or 200 nM miR-10a-5p inhibitor, and miR-10a-5p level was detected by qRT-PCR. (C) N2a cells pre-transfected with miR-10a-5p mimic, miR-10a-5p inhibitor, or miRNA NC were seeded in 96-well plates followed by PHEV infection. The cytopathic effect was monitored for 24 to 96 h. The virus titers were determined by TCID₅₀/ml and calculated according to the Reed–Muench method. (D) Cell Counting Kit- (CCK-8) assay was performed to determine the cell proliferation at the indicated times. (E) N2a cells were pre-transfected with 100 nM of miR-10a-5p mimic and then infected with PHEV at 2 × 10⁶ TCID₅₀/ml. The cells lystate were harvested at 24, 48, and 72 h post infection, and PHEV protein levels were determined by western blotting and normalized to beta actin. (F) Cells were treated as described in panel (E) and determined by qRT-PCR by analyzing PHEV mRNA level. GAPDH was chosen as a housekeeping gene for normalization. Data are shown as means ± SD of at least three independent experiments. *p < 0.05; ***p < 0.01.

FIGURE 3

Syndecan 1 (SDC1) expression was inhibited during porcine hemagglutinating encephalomyelitis virus (PHEV) infection. (A) N2a cells were incubated with PHEV for 24∼72 h in vitro, whereas the BALB/c mice were infected with PHEV for 3 or 5 days in vivo. N = 4 mice per group, three independent experiments. The endogenous SDC1 mRNA expression was determined by qRT-PCR. (B) The lysates from cells or mice that treated as described as (A) were harvested and detected by western blotting using primary antibodies against SDC1 or beta actin. (C) N2a cells were incubated with PHEV for 24 h and then fixed and labeled with antibodies against PHEV (green) and SDC1 (red). The cell nucleus was stained with DAPI (blue). Representative micrograph is shown, and quantitative analyses of the Rab3a staining are listed on the right. Bars indicate 20 μm. All of the data are representative of at least three independent experiments (*p < 0.05, **p < 0.01). (D) Sequence alignment of miR-10a-5p in TargetScan website and its target site in 3′ UTR of SDC1 mRNA are shown. Model of wild- and mutant-type constructs of SDC1 mRNA 3′ UTR. The red letters indicate the point mutation.

FIGURE 4

Syndecan 1 (SDC1) mRNA is a target of miR-10a-5p. (A) Wild-type (WT) or mutant-type (MUT) reporter constructs of SDC1 3’-UTR were co-transfected with 100 nM of miR-10a-5p mimic into HEK293T cells. After 24 h, the Renilla and firefly luciferase activities were assayed. (B) SDC1-WT or SDC1-MUT reporter was co-transfected with 200 nM of miR-10a-5p inhibitor into HEK293T cells for 24 h and were then harvested and subjected to luciferase activities detection. (C) N2a cells were transfected with different concentrations of miR-10a-5p mimic for 48 h, and the SDC1 mRNA expression was determined by qRT-PCR. (D) N2a cells pre-transfected with miR-10a-5p inhibitor for 48 h were subjected to SDC1 level determination by qRT-PCR. (E) Cells were pre-treated as indicated in panels (C,D), lysed, and detected by western blotting assay to examine the level of PHEV protein. Data were normalized to beta actin. All of the data are representative of at least three independent experiments (*p < 0.05, **p < 0.01). (F) Representative micrograph of IFA showed the expressed level of SDC1 (red) in the miR-10a-5p mimic- or mock-transfected N2a cells. Nucleus was stained with DAPI (blue). Quantitative analyses of the SDC1 staining are listed on the right. Bars, 20 μm.

FIGURE 5

Effects of Syndecan 1 (SDC1) defect in porcine hemagglutinating encephalomyelitis virus (PHEV) replication in vitro. (A) N2a cells were transfected with 50 nM of SDC1-siRNA-1, SDC1-siRNA-2, or negative control (NC) siRNA, and the expression of SDC1 mRNA was determined by qRT-PCR at 24 h post transfection. (B) The protein levels of SDC1 in the cell treated as described in panel (A) were determined by western blotting. (C) N2a cells were infected with PHEV for 24∼72 h, following SDC1-siRNA-1 transfection, and the levels of the viral genome RNA were detected by qRT-PCR. (D) Cells treated as indicated in panel (C) were harvested and determined by western blotting. (E) Viral titers in the supernatants were measured by TCID₅₀/ml assay. Data are shown as means ± SD of at least three independent experiments. *p < 0.05; **p < 0.001.

FIGURE 6

MiR-10a-5p agomir reduced porcine hemagglutinating encephalomyelitis virus (PHEV) replication in vivo. BALB/c mice were injected with miR-10a-5p agomir at 1 and 3 days post infection following PHEV infection. N = 6 mice per group, three independent experiments. The brain tissues were collected, and qRT-PCR and western blotting assay were performed to assess miR-10a-5p miRNA level (A), Syndecan 1 (SDC1) mRNA transcription (B), SDC1 protein expression (C), and viral replication and load (C,D). Phosphate-buffered saline (PBS)-injected mice were identified as mock injection, whereas mice in the control group were only infected with PHEV without agomir or PBS injection. (E) The titer of infectious virus in brain were measured by using a standard plaque assay in N2a cells. Data are shown as means ± SD of at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.