- Email: [email protected]
Altered expression of MiR-186-5p and its target genes after spinal cord ischemia–reperfusion injury in rats
Journal Pre-proof Altered Expression of MiR-186-5p and its Target Genes after Spinal Cord Ischemia–Reperfusion Injury in Rats Fengshou Chen, Xiaoqian Li, Zhe Li, Ziyun Qiang, Hong Ma
PII:
S0304-3940(19)30772-4
DOI:
https://doi.org/10.1016/j.neulet.2019.134669
Reference:
NSL 134669
To appear in:
Neuroscience Letters
Received Date:
11 June 2019
Revised Date:
21 October 2019
Accepted Date:
30 November 2019
Please cite this article as: Chen F, Li X, Li Z, Qiang Z, Ma H, Altered Expression of MiR-186-5p and its Target Genes after Spinal Cord Ischemia–Reperfusion Injury in Rats, Neuroscience Letters (2019), doi: https://doi.org/10.1016/j.neulet.2019.134669
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Altered Expression of MiR-186-5p and its Target Genes after Spinal Cord Ischemia–Reperfusion Injury in Rats
Fengshou Chen, Xiaoqian Li, Zhe Li, Ziyun Qiang，Hong Ma
Department of Anesthesiology, the First Hospital of China Medical University, Shenyang, Liaoning province, China
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Authors： 1) Fengshou Chen
Address: Department of Anesthesiology, the First Hospital of China
Medical University, Shenyang, Liaoning province, China.
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Email: [email protected]
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Tel: +86 24 83283100
Conflicts: Fengshou Chen reported no conflicts of interest.
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2) Xiaoqian Li
Address: Department of Anesthesiology, the First Hospital of China Medical University, Shenyang, Liaoning province, China.
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Email: [email protected] Tel: +86 24 83283100
3) Zhe Li
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Conflicts: Xiaoqian Li reported no conflicts of interest.
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Address: Department of Anesthesiology, the First Hospital of China Medical University, Shenyang, Liaoning province, China.
Email:[email protected] Tel: +86 24 83283100 Conflicts: Zhe Li reported no conflicts of interest.
4) Ziyun Qiang Address: Department of Anesthesiology, the First Hospital of China
Medical University, Shenyang, Liaoning province, China. Email:[email protected] Tel: +86 24 83283100 Conflicts: Ziyun Qiang reported no conflicts of interest. 5) Hong Ma Address: Department of Anesthesiology, the First Hospital of China Medical University, Shenyang, Liaoning province, China. Email: [email protected]
Conflicts: Hong MA reported no conflicts of interest.
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Tel: +86 24 83283100
Institution: Department of anesthesiology, the First Hospital of China Medical University.
*
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Corresponding author: Hong MA, MD, Ph.D.
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Address: No. 155 Nangjing North Street, Shenyang, Liaoning Province, China.
Address: Department of Anesthesiology, the First Affiliated Hospital of China
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Medical University, Shenyang, Liaoning province, China.
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Email: [email protected]
Highlights
Spinal cord ischemia–reperfusion (I/R) injury increased the expression of miR-186-5p. Mimic-186-5p improved neurological function and decreased cytokine expression. Wnt5a, TLR3, and CXCL13 increase after spinal cord I/R injury. Mimic-186-5p reduced the induction of Wnt5a, TLR3, and CXCL13.
ABSTRACT Spinal cord ischemia–reperfusion (I/R) injury remains an unresolved problem, and the
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mechanism is not fully elaborated. In a rat model of spinal cord I/R injury, we performed microarray analysis to examine the altered expression of microRNAs (miRs) at 24 h after the modelling. miR-186-5p was chosen for further study. An miR mimic or anti-miR
-p
oligonucleotide was intrathecally infused before the surgical procedure. The participation of miR-186-5p and its potential target genes based on bioinformatics analysis were analysed
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next. Pre-treatment with the miR-186-5p mimic improved neurological function and histological assessment scores; reduced Evans Blue extravasation; attenuated spinal cord
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oedema; and decreased interleukin 15 (IL-15), IL-6, IL-1β, and tumour necrosis factor-α (TNF-α) expression at 24 h after the modelling. KEGG analysis showed that the group of potential target genes of miR-186-5p was notably enriched in several signalling cascades,
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such as the Wnt, Hippo, and PI3K–AKT pathways. Gene Ontology (GO) analysis revealed that the group of potential target genes of miR-186-5p was significantly enriched in several
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biological processes, such as ‘Wnt signalling pathway’, ‘regulation of inflammatory response’, and ‘Toll-like receptor signalling pathway’. We further found that Wnt5a, TLR3, and
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chemokine (C-X-C motif) ligand 13 (CXCL13) were upregulated after the modelling and the miR-186-5p mimic reduced the induction of the aforementioned target genes. These data provide evidence that upregulation of miR-186-5p improves neurological outcomes induced by spinal cord I/R injury and may inhibit neuroinflammation through Wnt5a-, TLR3-, or CXCL13-mediated signal pathway in spinal cord I/R injury.
Keywords: MiR-186-5p, Spinal cord ischemia–reperfusion injury, Neuroinflammation, Wnt5a, TLR3, CXCL13
1. Introduction Thoracoabdominal aortic surgery presents the risk of spinal cord ischemia–reperfusion (I/R) injury[1]. Due to multifactorial pathogenic factors, spinal cord I/R injury still remains an unresolved problem, and the mechanism is not fully elaborated[2-5].
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MicroRNAs (miRNAs, miRs) are small oligonucleotides of noncoding RNA that base-pair with the 3-untranslated region of mRNA and regulate post-transcriptional expression[6]. Aberrant miRNA expression could contribute to many biological processes, including cellular differentiation and maintenance, neurogenesis, apoptosis, inflammation, and oxidative stress,
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during central nervous system (CNS) development and diseases[7]. miRNAs can affect their target genes involved in neuroprotection by inhibiting neuroinflammation, influencing
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autophagy, targeting antioxidant components, preventing apoptosis, and microglial
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activation[7-12]. However, there are only a few studies on the relation between miRNAs and spinal cord injury induced by I/R[13-16]. Nevertheless, the role of miR-186-5p has not been explored in spinal cord I/R injury. Here, in a rat model, we studied miR-186-5p and obtained
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further information regarding the miRNA function by bioinformatics analysis. 2. Material and Methods
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2.1. Experimental animals
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Experimental procedures were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Male Sprague–Dawley (SD) rats (200–250 g) were obtained from the Animal Center of China Medical University and maintained for 1 week prior to the surgical procedures. The Ethics Committee of China Medical University approved our experiment. 2.2. miRNA microarray analysis
At first, microarray analysis was performed to examine the altered expression of miRNAs. At 24 h after the modelling, we isolated miRNAs from segments L4–L6 of the spinal cord owing to their vulnerability to ischemic injury. We extracted total RNA samples using TRIzol Reagent (Thermo Fisher Scientific, USA) and conducted microarray analysis. 2.3. Quantification and time course of miR-186-5p expression after spinal cord I/R injury Fifty-six male SD rats were assigned by means of a random number table to seven groups: (1) sham group; (2) I/R 6 h group; (3) I/R 12 h group; (4) I/R 24 h group; (5) I/R 36 h group; (6)
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I/R 48 h group; and (7) I/R 72 h group. Rats were euthanized at 6, 12, 24, 36, 48, and 72 h after the modelling, respectively. Segments L4–L6 were collected. We extracted total RNA using TRIzol Reagent (Thermo Fisher Scientific, USA). cDNA was generated with the Prime Script® miRNA cDNA Synthesis Kit (TaKaRa, China)[13]. The levels of miR-186-5p were
The miR-186-5p-specific
primers
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determined using SYBR Premix qRT-PCR on a PCR System (Corbett Research, Australia). used
were
as
follows:
forward:
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5′-CCGCAAAGAATTCTCCTTTTGGGCT-3′ (Sanggo Biotech, China). U6 expression served as an internal control. We applied the 2ΔΔCt method to calculate data.
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2.4. Interventions
Forty male SD rats were assigned by means of a random number table to five groups: (1)
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sham group; (2) I/R group; (3) I/R + mimic-miR-186-5p group; (4) I/R + AMO-186-5p group; and (5) I/R + NC-miR-186-5p group. Rats were intrathecally injected with a miR mimic or oligonucleotide[13].
A
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anti-miR
synthetic
miR-186-5p
(5′-CAAAGAAUUCUCCUUUUGGGCU-3′),
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(5′-AGCCCAAAAGGAGAAUUCUUUG-3′),
mimic
AMO and
negative
(mimic-186-5p) (AMO-186-5p)
control
(NC-186-5p)
(5′-UUCUCCGAACGUGUCACGUTT-3′) were purchased from Jima Inc (China). Rats were intrathecally injected with liposome complexes of the oligonucleotides (50 mg/kg) and Lipofectamine® 2000 (Invitrogen, USA). Before the surgical procedure, rats were injected once a day for three consecutive days. 2.5. Rat model
As previously reported, a cross-clamped aortic arch was used to create the animal model[1, 13]. Pentobarbital sodium (50 mg/kg) was utilized for anesthetizing rats. Cross-clamping through the aorta was maintained for 14 min to create the rat model[17]. The same procedure without any block was performed on sham-operated rats. 2.6. Neurological evaluation At 24 h after the modelling, two observers evaluated hind limb function based on Tarlov scores: 0, no voluntary hind limb function; 1, poor hind limb motor function with perceptible
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movement; 2, joint motion present with no ability to stand; 3, stands and walks; and 4, normal hind limb function[1]. 2.7. Haematoxylin and eosin (H&E) staining
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At 24 h after the modelling, we collected and fixed segments L4–L6 in 4% paraformaldehyde. Then, we stained the tissues with H&E. Normal neurons of the stained sections were
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evaluated by two observers using a microscope (Leica, Germany). The appearance of basophilic stippling or diffused eosinophilic cytoplasm was used to identify normal or dead
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neurons [2, 4]. Intact neurons were calculated by averaging counts from three slides[4, 16]. 2.8. Evans blue (EB) extravasation
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EB extravasation into the parenchyma was used to determine blood–spinal cord barrier (BSCB) disruption[18]. At 24 h after the modelling, EB at a dose of 45 mg/kg was injected
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intravenously. After 1 h, we euthanized the rats. Segments L4–L6 were collected and homogenized with trichloroacetic acid, and the tissues were centrifuged. Supernatants were
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collected, and absorbance was measure at 632 nm using a spectrophotometer (TECAN, Austria). After fixing and sectioning (at 10-μm thickness) the samples, EB staining was recorded by fluorescent microscopy (Leica, German). 2.9. Water content of spinal cord At 24 h after the modelling, we euthanized the rats. Water content (%) was determined according to the formula: [(wet weight dry weight)/wet weight] × 100[19]. Segments L4–L6
were collected to obtain the wet weight. Then, the tissues were dried for 48 h at 105°C to calculate the dry weight. 2.10. ELISA At 24 h after the modelling, we collected segments L4–L6 to measure interleukin 15 (IL -15), IL-13, IL-6, IL-1β, and tumour necrosis factor-α (TNF-α) levels using ELISA kits purchased from Signalway Antibody Company (College Park, USA).
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2.11. Western blotting Protein expression levels in segments L4–L6 were detected by western blotting. Total proteins were extracted with RIPA buffer (KangChen, China). The antibodies used were rabbit polyclonal anti-IL-15 antibody (BioVision, USA), mouse monoclonal anti-IL-6
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(Abcam, USA), mouse monoclonal anti-IL-13 (Abcam, USA), rabbit polyclonal anti-IL-1β (Abcam, USA), rabbit polyclonal anti-TNFα (Abcam, USA), rabbit polyclonal anti-Wnt5a USA),
rabbit
polyclonal
anti-TLR3
(Bioss,
China),
rabbit
monoclonal
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(Abcam,
anti-chemokine (C-X-C motif) ligand 13 (CXCL13) (Abcam, USA), rabbit anti-GAPDH
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(Boster, China), and HRP-conjugated secondary antibodies (Beyotime , China). 2.12. Prediction of potential miR-186-5p target genes and bioinformatics analysis
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We predicted potential targets of miR-186-5p by means of the databases Miranda, PITA, miRWalk, and miRDB. Potential target genes were identified for miR-186-5p in ≥3 out of 4
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databases. Pathway analysis was performed in KEGG. Significance of functions of differentially expressed genes was determined by the 2 and Fisher’s exact tests at p < 0.05. A
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microRNA–pathway–gene network was built according to the relations of statistically significant pathways and genes as well as the relations among miR-186-5p, pathways, and genes. To determine biological attributes of the putative target genes and for identifying the biological functional coherence of the target genes, Gene Ontology (GO) analysis was performed on http://geneontology.org. We conducted multiple-comparison and Fisher’s exact test to calculate p-values and the false discovery rate of differentially expressed genes after retrieval of annotation functions of the miRNA targets.
2.13. Statistical analysis SPSS 15.0 (IBM, USA) was used for statistical analyses. The results were expressed as mean ± standard deviation. All variables measured in this study were normally distributed, and the groups were compared by Student’s t-test or one-way analysis of variance (ANOVA), followed by Newman–Keuls post-hoc analysis. p < 0.05 was defined as significant. 3. Results 3.1. Aberrant expression of miRNAs and expression of miR-186-5p We performed microarray analysis to examine the altered expression of miRNAs in the
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groups ‘I/R’ and ‘sham’. Five upregulated or downregulated miRNAs at 24 h after the modelling are presented in Table 1. Clustered heatmaps were created (Fig. 1A). Among these aberrantly expressed miRNAs, we chose miR-186-5p (p < 0.001) for further study.
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In addition, we studied the time course of miR-186-5p expression after the modelling. PCR analysis demonstrated that miR-186-5p levels decreased markedly at 12 (p = 0.007), 24 (p <
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0.001), and 36 h (p = 0.001) (Fig. 1B). At 24 h after the modelling, miR-186-5p expression reached the lowest level (p < 0.001; Fig. 1B). The data indicated that low miR-186-5p
modelling’ for further study.
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expression may correlate with I/R injury. We selected the time point ‘24 h after the
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3.2. Intrathecal pre-treatment with the miR-186-5p mimic or AMO successfully altered miR-186-5p expression
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Before the surgical procedure, mimic-186-5p, AMO-186-5p, or NC-186-5p were intrathecally injected. The injection was performed once a day for three consecutive days. At 24 h after the
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modelling, a synthetic miR mimic increased the expression of miR-186-5p significantly (p < 0.001). Intrathecal infusion of AMO-186-5p had the opposite effect (p < 0.001; Fig. 1C). 3.3. Intrathecal injection with the miR-186-5p mimic improved neurological function Fig. 1D demonstrates the neurological function of the five groups based on Tarlov scores after the modelling; each symbol symbolizes one rat. Spinal cord I/R injury caused severe neurological deficits of lower extremities (p < 0.001). Mimic-186-5p enhanced neurological
function recovery (p = 0.016). AMO-186-5p exacerbated neurological impairment (p = 0.040). 3.4. Intrathecal injection with the miR-186-5p mimic improved histologic evaluation Figs. 1E and F demonstrate histologic evaluation results at 24 h after ischemia. Intact neurons decreased significantly (p < 0.001). More intact neurons were found in operated rats subjected to mimic-186-5p injection (p < 0.001). AMO-186-5p injection induced more necrotic or dead neurons (p = 0.027).
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3.5. Intrathecal injection with the miR-186-5p mimic attenuated BSCB leakage
Extravasation of EB was observed in red under a fluorescent microscope[1, 4]. At 24 h after the modelling, EB extravasation in the I/R rats was higher (p < 0.001 versus sham-operated
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rats). In contrast, mimic-186-5p injection reduced extravasation (p < 0.001), but AMO-186-5p aggravated extravasation (p = 0.006), as depicted in Fig. 1F. Fluorescence density and content
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of EB were calculated (Fig. 1G and 1H). Mimic-186-5p injection reduced spinal cord oedema (p = 0.022), whereas intrathecal infusion of AMO-186-5p exacerbated spinal cord oedema (p
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= 0.041). Results are shown in Fig. 1I.
3.6. Intrathecal injection with the miR-186-5p mimic reduced IL-15, IL-6, IL-1β, and TNF-α
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levels
We assessed activation of IL-15, IL-6, IL-13, IL-1β, and TNF-α expression by ELISA after
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mimic-186-5p injection. The data indicated that IL-15, IL-6, IL-1β, and TNF-α were upregulated at 24 h after reperfusion (p < 0.0001). Mimic-186-5p injection significantly
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attenuated the induction of IL-15, IL-6, IL-1β, and TNF-α levels (p < 0.0001), whereas AMO-186-5p injection enhanced the upregulation of IL-15 (p < 0.0001), IL-6 (p < 0.0001), IL-1β (p < 0.0001), and TNF-α (p = 0.0008) (Fig. 2A–D). Expression of IL-13 manifested no differences among the five groups (data not shown). The expressions of IL-15, IL-6, IL-1β, and TNF-α were also measured by western blotting and demonstrated a similar trend (Fig. 2E–H). 3.7. Prediction of potential miR-186-5p targets and bioinformatics analysis
A total of 417 potential target genes were identified for miR-186-5p in ≥3 of four databases. Functional annotation of miR-186-5p was conducted by KEGG pathway analysis. The results were notably enriched in several pathways (p < 0.05), such as the Wnt signalling cascade, Hippo
signal
transduction,
and
PI3K–AKT
signalling
(Fig.
3A).
The
microRNA–pathway–gene network is illustrated in Fig. 3B. GO analysis revealed that the group of potential target genes of miR-186-5p was enriched significantly in several representative biological processes (p < 0.05), such as ‘apoptotic process’, ‘regulation of inflammatory response’, and ‘Toll-like receptor (TLR) signalling pathway’. The data are
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illustrated in Fig. 3C. It was noted that Wnt5a, TLR3, and CXCL13 were enriched in several GO functions (Table 2). Wnt5a, TLR3, and CXCL13 are associated with CNS neuroinflammation[20-23].We further confirmed the regulatory effects of mimic-186-5p on
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Wnt5a, TLR3, and CXCL13.
3.8. Effects of intrathecal miR-186-5p mimic injection on the expression of Wnt5a, TLR3,
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and CXCL13
The expression of Wnt5a, TLR3, and CXCL13 increased at 24 h after reperfusion (p < 0.0001,
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respectively). Intrathecal injection with mimic-186-5p significantly attenuated this upregulation (Wnt5a, p < 0.0001; TLR3, p = 0.0001; CXCL13, p < 0.0001), whereas
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AMO-186-5p injection enhanced the upregulation (Wnt5a, p = 0.0033; TLR3, p = 0.0020; CXCL13, p = 0.0339). The data are presented in Fig. 3D–F. The results indicated that Wnt5a, TLR3, and CXCL13 may be targets of miR-186-5p in spinal cord I/R injury in rats.
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4. Discussion
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Our research indicated that upregulation of miR-186-5p preserved hind limb function after spinal cord I/R injury (Fig. 1). Moreover, we demonstrated that increased miR-186-5p maintained intact neurons; attenuated BSCB leakage; and reduced the expression of IL-15, IL-6, IL-1β, and TNF-α after the modelling (Fig. 1 and 2). Based on bioinformatics analysis, we collected candidate target genes of miR-186-5p and found that Wnt5a, TLR3, and CXCL13 were regulated by miR-186-5p (Fig. 3). Therefore, these results suggested that upregulation of miR-186-5p ameliorated neurological outcomes induced by spinal I/R cord
injury
and
inhibited
neuroinflammation
possibly
through
Wnt5a-,
TLR3-,
or
CXCL13-mediated signal pathway in spinal cord I/R injury. Inflammation acts a vital factor in I/R injury[2, 17, 20]. Researches have proved that IL-5 participates in inflammatory responses in several diseases, such as chronic pancreatitis and Sjögren’s syndrome[24, 25]. Our results found that at 24 h after the modelling, IL-15 levels were elevated significantly due to I/R injury, whereas mimic-186-5p injection decreased the expression of IL-15 (Fig. 2). IL-6 performs a crucial function in inflammatory responses in the CNS[26]. A recent study has revealed that IL-6 increases the permeability of a vascular
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endothelial cell monolayer in vitro and inhibition of the effects of IL-6 reduces blood–brain barrier disruption after ischemia in ovine foetuses[27]. In the current study, expression of IL-6 manifested a trend similar to that of IL-15 and was decreased by intrathecal infusion of mimic-186-5p (Fig. 2). Previous studies have demonstrated that reduced production of IL-1β
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or TNF-α indicates a neuroprotective effect[28-30]. In the current study, IL-1β and TNF-α
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were reduced by mimic-186-5p injection at 24 h after the modelling (Fig. 2). In this study, pathway analysis implied that the group of potential target genes of miR-186-5p
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was notably enriched in several signalling cascades, such as Wnt, Hippo, and PI3K–AKT pathways (Fig. 3). Several studies have indicated that miR-186-5p is related to the
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above-mentioned pathways in other diseases[31, 32]. Overexpression of miR-186 inhibits Wnt signalling in retinoblastoma cells[31]. Upregulation of miR-186 leads to suppression of
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the Hippo signalling pathway in hepatocellular carcinoma cells[32]. GO analysis suggested that Wnt5a, TLR3, and CXCL13 were enriched in several GO
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functions (Table 2), and thus, we explored the effects of mimic-186-5p on the expression of Wnt5a, TLR3, and CXCL13 (Fig. 3). Wnt5a plays an important role in a variety of inflammatory disorders, such as rheumatoid arthritis, atherosclerosis, and sepsis[33]. Li B et al. demonstrated that activation of Wnt5a signalling elicits the expression of proinflammatory cytokines IL-1β and TNF-α, whereas inhibition of Wnt5a signalling has the reverse effect[22]. Wnt5a-regulated neuroinflammation may contribute to the development of chronic pain in aging mice, and inhibition of Wnt5a blocks 2′-3′-dideoxycytidine-induced upregulation of
TNF-α and astrocyte reaction[21]. In this study, we found significant change in the expression of Wnt5a after the modelling (Fig. 3), which may be associated with the release of cytokines and astrocyte reaction. Intrathecal injection with mimic-186-5p significantly attenuated the upregulation of Wnt5a (Fig. 3) Previous research has revealed that TLR3 is regulated by miR-186-5p in cardiomyocytes treated with a high glucose concentration[34]. TLR3 is involved in spinal cord microglia activation and neuropathic pain[35, 36]. TLR3–cytokine pathway has been proven to play a role in neuroinflammation during spinal cord I/R injury[20]. We found that intrathecal injection with mimic-186-5p significantly decreased the
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expression of TLR3 (Fig. 3), implying that miR-186-5p may regulate neuroinflammation during spinal cord I/R injury via regulating the TLR3–cytokine pathway. Recent data have shown induction of CXCL13 under a variety of circumstances during CNS inflammation, such as HIV encephalopathy, neurosyphilis, and Lyme neuroborreliosis[23]. CXCL13 is
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regulated by miR-186-5p in neuropathic pain[37]. In this study, we found that CXCL13 was induced by spinal cord I/R injury and that intrathecal injection with mimic-186-5p
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significantly reduced the induction of CXCL13 (Fig. 3). CXCL13 may be the target gene of miR-186-5p for regulating neuroinflammation after the modelling.
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5. Conclusion
In conclusion, we demonstrated that upregulation of miR-186-5p preserved hind limb
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function after spinal cord I/R injury by maintaining intact motor neurons; attenuating BSCB leakage; and reducing expression of IL-15, IL-6, IL-1β, and TNF-α. We speculated that inhibited
neuroinflammation
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miR-186-5p
possibly
through
Wnt5a-,
TLR3-,
or
CXCL13-mediated signal pathway in spinal cord I/R injury. miR-186-5p may be a potential
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clinical target for inhibiting neuroinflammation in spinal cord I/R injury.
Acknowledgements This work is supported by the Natural Science Foundation of China (grant number 81771342).
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The Journal of clinical investigation 126 (2016) 745-761.
Figure Caption
Fig. 1. Aberrant expression of miR-186-5p and effects of mimic-186-5p at 24 h after the modelling. (A) Hierarchical clustering analyses concerning the selected differentially expressed miRs in groups ‘sham’ and ‘I/R’ (n = 3, respectively). Red colour signifies high expression; green signifies low expression. Each column symbolizes one sample. (B) Time course of miR-186-5p relative expression. (C) Expression of miR-186-5p in five groups at 24 h after the modelling. (D) Tarlov scores. (E) Number of intact neurons. (F) Representative
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H&E staining images and fluorescence images of extravasating EB. (G) Fluorescence density of EB (INT/mm2). (H) Spinal cord EB content (μg/g). (I) Percentage water content. *p < 0.05, versus group sham, @p < 0.05 versus group I/R 24 h, #p < 0.05 versus group I/R.
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Fig. 2. Effects of mimic-186-5p and AMO-186-5p on the levels of IL-15, IL-6, IL-1β, and TNF-α evaluated by ELISA (A–D) and western blotting (E–H) at 24 h after the modelling. *p
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< 0.05 versus group sham, #p < 0.05 versus group I/R.
Fig. 3. Bioinformatics analysis of potential rno-miR-186-5p target mRNAs (i.e., genes). (A)
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Representative KEGG pathway terms. (B) The miR–pathway–gene network. (C) Representative biological process terms. Effects of mimic-186-5p and AMO-186-5p on the protein expression of Wnt5a (D), TLR3 (E), and CXCL13 (F) at 24 h after the modelling. p <
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versus group I/R.
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0.05 for all representative GO and pathway terms. *p < 0.05 versus group sham, #p < 0.05
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Table 1 Five upregulated or downregulated miRNAs in the spinal cord of rats at 24 h after I/R injury compared with those in sham-operated rats (n = 3 per group) Fold change
P-value
rno-miR-186-5p
P-value
rno-miR-144-3 0.27492
0.0002
p
2.55371
0.0126
0.46886
0.0114
rno-miR-3568
3.57657
0.0194
rno-miR-291a-3p
rno-miR-30b-3 0.34972
0.0039
rno-miR-702-3p
p
9.81339
0.0295
3.40141
0.0005
2.6917
0.0034
rno-miR-423-5 0.48282
0.0436
rno-miR-365-3p
p
rno-miR-300-5
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p
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0.0327
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0.45454
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Fold change
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rno-miR-129-1-3p
miRNAs
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miRNAs
Table 2 Representative 20 GO terms with significantly enriched function for potential targets of miR-186-5p Go Name
Gene
cell surface receptor signaling pathway
Adgrg1, Cd24, Tshr, Glp1r, Calcr, Cblb, Adgrl4, Tspan13, Mapk6, Cxcl13, Mapkapk3 Etv5, Ephb4, Rmdn3, Fgfr2, Mitf, Purb, Ets1, Nr1d2, Wnt5a, Ehf, Gli2, Elk4, Piwil4, Spast, Epha4, Etv1
cell differentiation regulation of gene expression cAMP-mediated signaling
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Six1, Atxn7l3b, LOC103689943, Hey2, Mapk6, Mitf, Tob1, Sort1, Ddx3x, Tsku, Tbl1xr1, Marf1, Prkaa2 Glp1r, Lep, Ndufs4, Adcyap1, Rims2
Ank3, Dlc1, Prom2, Snx27, Gja1, Cxxc5, Cblb, Lrrc28, Depdc1b, Unc5c, Arap2, Sall1, Gabrr1, Trhde, Dcdc2, Wisp3, Ptprm, Agap1 Bnip3, Asah2, Six1, Unc5c, Casp9, Mfn2, Gja1, Rps6ka3, Rmdn3, Plagl2, Dlc1, Rnf152
apoptotic process
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signal transduction
Wnt signaling pathway, planar cell polarity pathway cell-cell signaling
Cd24, Cxcl13, Gja1, Wisp3, Bhlha15, Fgfr2, Trhde
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positive regulation of apoptotic process
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Vangl2, Wnt9b, Wnt5a
Ddx3x, Tlr3, Unc5c, Tnfsf10, Phlda3, Itga6, Camk1d, Casp9, Tomm22, Tnfrsf8, Bnip3
central nervous system
Map2, Btg2, Lep
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neuron development
positive regulation of ERK1 and ERK2 cascade
Dstyk, Adcyap1, Prkca, Epha4, Sema7a, Calcr, Dnajc27, Fgfr2, Ephb4
negative regulation of
Prkca, Nf2, Chp1, Lrtm2, Lrrtm3, Ubash3b protein kinase activity
regulation of cell proliferation
Brca1, Pds5b, Fgfr2, Cxcl13, Prdm1, Tnfrsf8, Nf2, Sat1, Mitf
toll-like receptor signaling
Mapkapk3, Rps6ka3, Tlr3 pathway intrinsic apoptotic signaling
Bnip3, Ddx3x, Prkca pathway regulation of Wnt signaling
Mdfic, Vangl2, Dcdc2
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pathway neuron migration
Usp9x, Gja1, Neurod4, Dcdc2, Dclk1, Dcx
neuron differentiation
Wnt9b, Wnt5a, Mtpn, Cops2, Btg2, Atp2b2, Hipk1
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toll-like receptor 3 signaling
Tlr3, Scara3
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pathway regulation of inflammatory
Nr1d2, Adamts12, Brd4, Sema7a
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response
PII:
S0304-3940(19)30772-4
DOI:
https://doi.org/10.1016/j.neulet.2019.134669
Reference:
NSL 134669
To appear in:
Neuroscience Letters
Received Date:
11 June 2019
Revised Date:
21 October 2019
Accepted Date:
30 November 2019
Please cite this article as: Chen F, Li X, Li Z, Qiang Z, Ma H, Altered Expression of MiR-186-5p and its Target Genes after Spinal Cord Ischemia–Reperfusion Injury in Rats, Neuroscience Letters (2019), doi: https://doi.org/10.1016/j.neulet.2019.134669
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Altered Expression of MiR-186-5p and its Target Genes after Spinal Cord Ischemia–Reperfusion Injury in Rats
Fengshou Chen, Xiaoqian Li, Zhe Li, Ziyun Qiang，Hong Ma
Department of Anesthesiology, the First Hospital of China Medical University, Shenyang, Liaoning province, China
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Authors： 1) Fengshou Chen
Address: Department of Anesthesiology, the First Hospital of China
Medical University, Shenyang, Liaoning province, China.
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Email: [email protected]
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Tel: +86 24 83283100
Conflicts: Fengshou Chen reported no conflicts of interest.
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2) Xiaoqian Li
Address: Department of Anesthesiology, the First Hospital of China Medical University, Shenyang, Liaoning province, China.
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Email: [email protected] Tel: +86 24 83283100
3) Zhe Li
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Conflicts: Xiaoqian Li reported no conflicts of interest.
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Address: Department of Anesthesiology, the First Hospital of China Medical University, Shenyang, Liaoning province, China.
Email:[email protected] Tel: +86 24 83283100 Conflicts: Zhe Li reported no conflicts of interest.
4) Ziyun Qiang Address: Department of Anesthesiology, the First Hospital of China
Medical University, Shenyang, Liaoning province, China. Email:[email protected] Tel: +86 24 83283100 Conflicts: Ziyun Qiang reported no conflicts of interest. 5) Hong Ma Address: Department of Anesthesiology, the First Hospital of China Medical University, Shenyang, Liaoning province, China. Email: [email protected]
Conflicts: Hong MA reported no conflicts of interest.
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Tel: +86 24 83283100
Institution: Department of anesthesiology, the First Hospital of China Medical University.
*
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Corresponding author: Hong MA, MD, Ph.D.
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Address: No. 155 Nangjing North Street, Shenyang, Liaoning Province, China.
Address: Department of Anesthesiology, the First Affiliated Hospital of China
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Medical University, Shenyang, Liaoning province, China.
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Email: [email protected]
Highlights
Spinal cord ischemia–reperfusion (I/R) injury increased the expression of miR-186-5p. Mimic-186-5p improved neurological function and decreased cytokine expression. Wnt5a, TLR3, and CXCL13 increase after spinal cord I/R injury. Mimic-186-5p reduced the induction of Wnt5a, TLR3, and CXCL13.
ABSTRACT Spinal cord ischemia–reperfusion (I/R) injury remains an unresolved problem, and the
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mechanism is not fully elaborated. In a rat model of spinal cord I/R injury, we performed microarray analysis to examine the altered expression of microRNAs (miRs) at 24 h after the modelling. miR-186-5p was chosen for further study. An miR mimic or anti-miR
-p
oligonucleotide was intrathecally infused before the surgical procedure. The participation of miR-186-5p and its potential target genes based on bioinformatics analysis were analysed
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next. Pre-treatment with the miR-186-5p mimic improved neurological function and histological assessment scores; reduced Evans Blue extravasation; attenuated spinal cord
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oedema; and decreased interleukin 15 (IL-15), IL-6, IL-1β, and tumour necrosis factor-α (TNF-α) expression at 24 h after the modelling. KEGG analysis showed that the group of potential target genes of miR-186-5p was notably enriched in several signalling cascades,
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such as the Wnt, Hippo, and PI3K–AKT pathways. Gene Ontology (GO) analysis revealed that the group of potential target genes of miR-186-5p was significantly enriched in several
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biological processes, such as ‘Wnt signalling pathway’, ‘regulation of inflammatory response’, and ‘Toll-like receptor signalling pathway’. We further found that Wnt5a, TLR3, and
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chemokine (C-X-C motif) ligand 13 (CXCL13) were upregulated after the modelling and the miR-186-5p mimic reduced the induction of the aforementioned target genes. These data provide evidence that upregulation of miR-186-5p improves neurological outcomes induced by spinal cord I/R injury and may inhibit neuroinflammation through Wnt5a-, TLR3-, or CXCL13-mediated signal pathway in spinal cord I/R injury.
Keywords: MiR-186-5p, Spinal cord ischemia–reperfusion injury, Neuroinflammation, Wnt5a, TLR3, CXCL13
1. Introduction Thoracoabdominal aortic surgery presents the risk of spinal cord ischemia–reperfusion (I/R) injury[1]. Due to multifactorial pathogenic factors, spinal cord I/R injury still remains an unresolved problem, and the mechanism is not fully elaborated[2-5].
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MicroRNAs (miRNAs, miRs) are small oligonucleotides of noncoding RNA that base-pair with the 3-untranslated region of mRNA and regulate post-transcriptional expression[6]. Aberrant miRNA expression could contribute to many biological processes, including cellular differentiation and maintenance, neurogenesis, apoptosis, inflammation, and oxidative stress,
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during central nervous system (CNS) development and diseases[7]. miRNAs can affect their target genes involved in neuroprotection by inhibiting neuroinflammation, influencing
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autophagy, targeting antioxidant components, preventing apoptosis, and microglial
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activation[7-12]. However, there are only a few studies on the relation between miRNAs and spinal cord injury induced by I/R[13-16]. Nevertheless, the role of miR-186-5p has not been explored in spinal cord I/R injury. Here, in a rat model, we studied miR-186-5p and obtained
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further information regarding the miRNA function by bioinformatics analysis. 2. Material and Methods
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2.1. Experimental animals
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Experimental procedures were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Male Sprague–Dawley (SD) rats (200–250 g) were obtained from the Animal Center of China Medical University and maintained for 1 week prior to the surgical procedures. The Ethics Committee of China Medical University approved our experiment. 2.2. miRNA microarray analysis
At first, microarray analysis was performed to examine the altered expression of miRNAs. At 24 h after the modelling, we isolated miRNAs from segments L4–L6 of the spinal cord owing to their vulnerability to ischemic injury. We extracted total RNA samples using TRIzol Reagent (Thermo Fisher Scientific, USA) and conducted microarray analysis. 2.3. Quantification and time course of miR-186-5p expression after spinal cord I/R injury Fifty-six male SD rats were assigned by means of a random number table to seven groups: (1) sham group; (2) I/R 6 h group; (3) I/R 12 h group; (4) I/R 24 h group; (5) I/R 36 h group; (6)
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I/R 48 h group; and (7) I/R 72 h group. Rats were euthanized at 6, 12, 24, 36, 48, and 72 h after the modelling, respectively. Segments L4–L6 were collected. We extracted total RNA using TRIzol Reagent (Thermo Fisher Scientific, USA). cDNA was generated with the Prime Script® miRNA cDNA Synthesis Kit (TaKaRa, China)[13]. The levels of miR-186-5p were
The miR-186-5p-specific
primers
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determined using SYBR Premix qRT-PCR on a PCR System (Corbett Research, Australia). used
were
as
follows:
forward:
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5′-CCGCAAAGAATTCTCCTTTTGGGCT-3′ (Sanggo Biotech, China). U6 expression served as an internal control. We applied the 2ΔΔCt method to calculate data.
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2.4. Interventions
Forty male SD rats were assigned by means of a random number table to five groups: (1)
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sham group; (2) I/R group; (3) I/R + mimic-miR-186-5p group; (4) I/R + AMO-186-5p group; and (5) I/R + NC-miR-186-5p group. Rats were intrathecally injected with a miR mimic or oligonucleotide[13].
A
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anti-miR
synthetic
miR-186-5p
(5′-CAAAGAAUUCUCCUUUUGGGCU-3′),
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(5′-AGCCCAAAAGGAGAAUUCUUUG-3′),
mimic
AMO and
negative
(mimic-186-5p) (AMO-186-5p)
control
(NC-186-5p)
(5′-UUCUCCGAACGUGUCACGUTT-3′) were purchased from Jima Inc (China). Rats were intrathecally injected with liposome complexes of the oligonucleotides (50 mg/kg) and Lipofectamine® 2000 (Invitrogen, USA). Before the surgical procedure, rats were injected once a day for three consecutive days. 2.5. Rat model
As previously reported, a cross-clamped aortic arch was used to create the animal model[1, 13]. Pentobarbital sodium (50 mg/kg) was utilized for anesthetizing rats. Cross-clamping through the aorta was maintained for 14 min to create the rat model[17]. The same procedure without any block was performed on sham-operated rats. 2.6. Neurological evaluation At 24 h after the modelling, two observers evaluated hind limb function based on Tarlov scores: 0, no voluntary hind limb function; 1, poor hind limb motor function with perceptible
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movement; 2, joint motion present with no ability to stand; 3, stands and walks; and 4, normal hind limb function[1]. 2.7. Haematoxylin and eosin (H&E) staining
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At 24 h after the modelling, we collected and fixed segments L4–L6 in 4% paraformaldehyde. Then, we stained the tissues with H&E. Normal neurons of the stained sections were
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evaluated by two observers using a microscope (Leica, Germany). The appearance of basophilic stippling or diffused eosinophilic cytoplasm was used to identify normal or dead
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neurons [2, 4]. Intact neurons were calculated by averaging counts from three slides[4, 16]. 2.8. Evans blue (EB) extravasation
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EB extravasation into the parenchyma was used to determine blood–spinal cord barrier (BSCB) disruption[18]. At 24 h after the modelling, EB at a dose of 45 mg/kg was injected
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intravenously. After 1 h, we euthanized the rats. Segments L4–L6 were collected and homogenized with trichloroacetic acid, and the tissues were centrifuged. Supernatants were
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collected, and absorbance was measure at 632 nm using a spectrophotometer (TECAN, Austria). After fixing and sectioning (at 10-μm thickness) the samples, EB staining was recorded by fluorescent microscopy (Leica, German). 2.9. Water content of spinal cord At 24 h after the modelling, we euthanized the rats. Water content (%) was determined according to the formula: [(wet weight dry weight)/wet weight] × 100[19]. Segments L4–L6
were collected to obtain the wet weight. Then, the tissues were dried for 48 h at 105°C to calculate the dry weight. 2.10. ELISA At 24 h after the modelling, we collected segments L4–L6 to measure interleukin 15 (IL -15), IL-13, IL-6, IL-1β, and tumour necrosis factor-α (TNF-α) levels using ELISA kits purchased from Signalway Antibody Company (College Park, USA).
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2.11. Western blotting Protein expression levels in segments L4–L6 were detected by western blotting. Total proteins were extracted with RIPA buffer (KangChen, China). The antibodies used were rabbit polyclonal anti-IL-15 antibody (BioVision, USA), mouse monoclonal anti-IL-6
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(Abcam, USA), mouse monoclonal anti-IL-13 (Abcam, USA), rabbit polyclonal anti-IL-1β (Abcam, USA), rabbit polyclonal anti-TNFα (Abcam, USA), rabbit polyclonal anti-Wnt5a USA),
rabbit
polyclonal
anti-TLR3
(Bioss,
China),
rabbit
monoclonal
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(Abcam,
anti-chemokine (C-X-C motif) ligand 13 (CXCL13) (Abcam, USA), rabbit anti-GAPDH
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(Boster, China), and HRP-conjugated secondary antibodies (Beyotime , China). 2.12. Prediction of potential miR-186-5p target genes and bioinformatics analysis
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We predicted potential targets of miR-186-5p by means of the databases Miranda, PITA, miRWalk, and miRDB. Potential target genes were identified for miR-186-5p in ≥3 out of 4
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databases. Pathway analysis was performed in KEGG. Significance of functions of differentially expressed genes was determined by the 2 and Fisher’s exact tests at p < 0.05. A
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microRNA–pathway–gene network was built according to the relations of statistically significant pathways and genes as well as the relations among miR-186-5p, pathways, and genes. To determine biological attributes of the putative target genes and for identifying the biological functional coherence of the target genes, Gene Ontology (GO) analysis was performed on http://geneontology.org. We conducted multiple-comparison and Fisher’s exact test to calculate p-values and the false discovery rate of differentially expressed genes after retrieval of annotation functions of the miRNA targets.
2.13. Statistical analysis SPSS 15.0 (IBM, USA) was used for statistical analyses. The results were expressed as mean ± standard deviation. All variables measured in this study were normally distributed, and the groups were compared by Student’s t-test or one-way analysis of variance (ANOVA), followed by Newman–Keuls post-hoc analysis. p < 0.05 was defined as significant. 3. Results 3.1. Aberrant expression of miRNAs and expression of miR-186-5p We performed microarray analysis to examine the altered expression of miRNAs in the
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groups ‘I/R’ and ‘sham’. Five upregulated or downregulated miRNAs at 24 h after the modelling are presented in Table 1. Clustered heatmaps were created (Fig. 1A). Among these aberrantly expressed miRNAs, we chose miR-186-5p (p < 0.001) for further study.
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In addition, we studied the time course of miR-186-5p expression after the modelling. PCR analysis demonstrated that miR-186-5p levels decreased markedly at 12 (p = 0.007), 24 (p <
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0.001), and 36 h (p = 0.001) (Fig. 1B). At 24 h after the modelling, miR-186-5p expression reached the lowest level (p < 0.001; Fig. 1B). The data indicated that low miR-186-5p
modelling’ for further study.
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expression may correlate with I/R injury. We selected the time point ‘24 h after the
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3.2. Intrathecal pre-treatment with the miR-186-5p mimic or AMO successfully altered miR-186-5p expression
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Before the surgical procedure, mimic-186-5p, AMO-186-5p, or NC-186-5p were intrathecally injected. The injection was performed once a day for three consecutive days. At 24 h after the
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modelling, a synthetic miR mimic increased the expression of miR-186-5p significantly (p < 0.001). Intrathecal infusion of AMO-186-5p had the opposite effect (p < 0.001; Fig. 1C). 3.3. Intrathecal injection with the miR-186-5p mimic improved neurological function Fig. 1D demonstrates the neurological function of the five groups based on Tarlov scores after the modelling; each symbol symbolizes one rat. Spinal cord I/R injury caused severe neurological deficits of lower extremities (p < 0.001). Mimic-186-5p enhanced neurological
function recovery (p = 0.016). AMO-186-5p exacerbated neurological impairment (p = 0.040). 3.4. Intrathecal injection with the miR-186-5p mimic improved histologic evaluation Figs. 1E and F demonstrate histologic evaluation results at 24 h after ischemia. Intact neurons decreased significantly (p < 0.001). More intact neurons were found in operated rats subjected to mimic-186-5p injection (p < 0.001). AMO-186-5p injection induced more necrotic or dead neurons (p = 0.027).
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3.5. Intrathecal injection with the miR-186-5p mimic attenuated BSCB leakage
Extravasation of EB was observed in red under a fluorescent microscope[1, 4]. At 24 h after the modelling, EB extravasation in the I/R rats was higher (p < 0.001 versus sham-operated
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rats). In contrast, mimic-186-5p injection reduced extravasation (p < 0.001), but AMO-186-5p aggravated extravasation (p = 0.006), as depicted in Fig. 1F. Fluorescence density and content
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of EB were calculated (Fig. 1G and 1H). Mimic-186-5p injection reduced spinal cord oedema (p = 0.022), whereas intrathecal infusion of AMO-186-5p exacerbated spinal cord oedema (p
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= 0.041). Results are shown in Fig. 1I.
3.6. Intrathecal injection with the miR-186-5p mimic reduced IL-15, IL-6, IL-1β, and TNF-α
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levels
We assessed activation of IL-15, IL-6, IL-13, IL-1β, and TNF-α expression by ELISA after
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mimic-186-5p injection. The data indicated that IL-15, IL-6, IL-1β, and TNF-α were upregulated at 24 h after reperfusion (p < 0.0001). Mimic-186-5p injection significantly
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attenuated the induction of IL-15, IL-6, IL-1β, and TNF-α levels (p < 0.0001), whereas AMO-186-5p injection enhanced the upregulation of IL-15 (p < 0.0001), IL-6 (p < 0.0001), IL-1β (p < 0.0001), and TNF-α (p = 0.0008) (Fig. 2A–D). Expression of IL-13 manifested no differences among the five groups (data not shown). The expressions of IL-15, IL-6, IL-1β, and TNF-α were also measured by western blotting and demonstrated a similar trend (Fig. 2E–H). 3.7. Prediction of potential miR-186-5p targets and bioinformatics analysis
A total of 417 potential target genes were identified for miR-186-5p in ≥3 of four databases. Functional annotation of miR-186-5p was conducted by KEGG pathway analysis. The results were notably enriched in several pathways (p < 0.05), such as the Wnt signalling cascade, Hippo
signal
transduction,
and
PI3K–AKT
signalling
(Fig.
3A).
The
microRNA–pathway–gene network is illustrated in Fig. 3B. GO analysis revealed that the group of potential target genes of miR-186-5p was enriched significantly in several representative biological processes (p < 0.05), such as ‘apoptotic process’, ‘regulation of inflammatory response’, and ‘Toll-like receptor (TLR) signalling pathway’. The data are
ro of
illustrated in Fig. 3C. It was noted that Wnt5a, TLR3, and CXCL13 were enriched in several GO functions (Table 2). Wnt5a, TLR3, and CXCL13 are associated with CNS neuroinflammation[20-23].We further confirmed the regulatory effects of mimic-186-5p on
-p
Wnt5a, TLR3, and CXCL13.
3.8. Effects of intrathecal miR-186-5p mimic injection on the expression of Wnt5a, TLR3,
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and CXCL13
The expression of Wnt5a, TLR3, and CXCL13 increased at 24 h after reperfusion (p < 0.0001,
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respectively). Intrathecal injection with mimic-186-5p significantly attenuated this upregulation (Wnt5a, p < 0.0001; TLR3, p = 0.0001; CXCL13, p < 0.0001), whereas
na
AMO-186-5p injection enhanced the upregulation (Wnt5a, p = 0.0033; TLR3, p = 0.0020; CXCL13, p = 0.0339). The data are presented in Fig. 3D–F. The results indicated that Wnt5a, TLR3, and CXCL13 may be targets of miR-186-5p in spinal cord I/R injury in rats.
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4. Discussion
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Our research indicated that upregulation of miR-186-5p preserved hind limb function after spinal cord I/R injury (Fig. 1). Moreover, we demonstrated that increased miR-186-5p maintained intact neurons; attenuated BSCB leakage; and reduced the expression of IL-15, IL-6, IL-1β, and TNF-α after the modelling (Fig. 1 and 2). Based on bioinformatics analysis, we collected candidate target genes of miR-186-5p and found that Wnt5a, TLR3, and CXCL13 were regulated by miR-186-5p (Fig. 3). Therefore, these results suggested that upregulation of miR-186-5p ameliorated neurological outcomes induced by spinal I/R cord
injury
and
inhibited
neuroinflammation
possibly
through
Wnt5a-,
TLR3-,
or
CXCL13-mediated signal pathway in spinal cord I/R injury. Inflammation acts a vital factor in I/R injury[2, 17, 20]. Researches have proved that IL-5 participates in inflammatory responses in several diseases, such as chronic pancreatitis and Sjögren’s syndrome[24, 25]. Our results found that at 24 h after the modelling, IL-15 levels were elevated significantly due to I/R injury, whereas mimic-186-5p injection decreased the expression of IL-15 (Fig. 2). IL-6 performs a crucial function in inflammatory responses in the CNS[26]. A recent study has revealed that IL-6 increases the permeability of a vascular
ro of
endothelial cell monolayer in vitro and inhibition of the effects of IL-6 reduces blood–brain barrier disruption after ischemia in ovine foetuses[27]. In the current study, expression of IL-6 manifested a trend similar to that of IL-15 and was decreased by intrathecal infusion of mimic-186-5p (Fig. 2). Previous studies have demonstrated that reduced production of IL-1β
-p
or TNF-α indicates a neuroprotective effect[28-30]. In the current study, IL-1β and TNF-α
re
were reduced by mimic-186-5p injection at 24 h after the modelling (Fig. 2). In this study, pathway analysis implied that the group of potential target genes of miR-186-5p
lP
was notably enriched in several signalling cascades, such as Wnt, Hippo, and PI3K–AKT pathways (Fig. 3). Several studies have indicated that miR-186-5p is related to the
na
above-mentioned pathways in other diseases[31, 32]. Overexpression of miR-186 inhibits Wnt signalling in retinoblastoma cells[31]. Upregulation of miR-186 leads to suppression of
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the Hippo signalling pathway in hepatocellular carcinoma cells[32]. GO analysis suggested that Wnt5a, TLR3, and CXCL13 were enriched in several GO
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functions (Table 2), and thus, we explored the effects of mimic-186-5p on the expression of Wnt5a, TLR3, and CXCL13 (Fig. 3). Wnt5a plays an important role in a variety of inflammatory disorders, such as rheumatoid arthritis, atherosclerosis, and sepsis[33]. Li B et al. demonstrated that activation of Wnt5a signalling elicits the expression of proinflammatory cytokines IL-1β and TNF-α, whereas inhibition of Wnt5a signalling has the reverse effect[22]. Wnt5a-regulated neuroinflammation may contribute to the development of chronic pain in aging mice, and inhibition of Wnt5a blocks 2′-3′-dideoxycytidine-induced upregulation of
TNF-α and astrocyte reaction[21]. In this study, we found significant change in the expression of Wnt5a after the modelling (Fig. 3), which may be associated with the release of cytokines and astrocyte reaction. Intrathecal injection with mimic-186-5p significantly attenuated the upregulation of Wnt5a (Fig. 3) Previous research has revealed that TLR3 is regulated by miR-186-5p in cardiomyocytes treated with a high glucose concentration[34]. TLR3 is involved in spinal cord microglia activation and neuropathic pain[35, 36]. TLR3–cytokine pathway has been proven to play a role in neuroinflammation during spinal cord I/R injury[20]. We found that intrathecal injection with mimic-186-5p significantly decreased the
ro of
expression of TLR3 (Fig. 3), implying that miR-186-5p may regulate neuroinflammation during spinal cord I/R injury via regulating the TLR3–cytokine pathway. Recent data have shown induction of CXCL13 under a variety of circumstances during CNS inflammation, such as HIV encephalopathy, neurosyphilis, and Lyme neuroborreliosis[23]. CXCL13 is
-p
regulated by miR-186-5p in neuropathic pain[37]. In this study, we found that CXCL13 was induced by spinal cord I/R injury and that intrathecal injection with mimic-186-5p
re
significantly reduced the induction of CXCL13 (Fig. 3). CXCL13 may be the target gene of miR-186-5p for regulating neuroinflammation after the modelling.
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5. Conclusion
In conclusion, we demonstrated that upregulation of miR-186-5p preserved hind limb
na
function after spinal cord I/R injury by maintaining intact motor neurons; attenuating BSCB leakage; and reducing expression of IL-15, IL-6, IL-1β, and TNF-α. We speculated that inhibited
neuroinflammation
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miR-186-5p
possibly
through
Wnt5a-,
TLR3-,
or
CXCL13-mediated signal pathway in spinal cord I/R injury. miR-186-5p may be a potential
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clinical target for inhibiting neuroinflammation in spinal cord I/R injury.
Acknowledgements This work is supported by the Natural Science Foundation of China (grant number 81771342).
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Figure Caption
Fig. 1. Aberrant expression of miR-186-5p and effects of mimic-186-5p at 24 h after the modelling. (A) Hierarchical clustering analyses concerning the selected differentially expressed miRs in groups ‘sham’ and ‘I/R’ (n = 3, respectively). Red colour signifies high expression; green signifies low expression. Each column symbolizes one sample. (B) Time course of miR-186-5p relative expression. (C) Expression of miR-186-5p in five groups at 24 h after the modelling. (D) Tarlov scores. (E) Number of intact neurons. (F) Representative
ro of
H&E staining images and fluorescence images of extravasating EB. (G) Fluorescence density of EB (INT/mm2). (H) Spinal cord EB content (μg/g). (I) Percentage water content. *p < 0.05, versus group sham, @p < 0.05 versus group I/R 24 h, #p < 0.05 versus group I/R.
-p
Fig. 2. Effects of mimic-186-5p and AMO-186-5p on the levels of IL-15, IL-6, IL-1β, and TNF-α evaluated by ELISA (A–D) and western blotting (E–H) at 24 h after the modelling. *p
re
< 0.05 versus group sham, #p < 0.05 versus group I/R.
Fig. 3. Bioinformatics analysis of potential rno-miR-186-5p target mRNAs (i.e., genes). (A)
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Representative KEGG pathway terms. (B) The miR–pathway–gene network. (C) Representative biological process terms. Effects of mimic-186-5p and AMO-186-5p on the protein expression of Wnt5a (D), TLR3 (E), and CXCL13 (F) at 24 h after the modelling. p <
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versus group I/R.
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0.05 for all representative GO and pathway terms. *p < 0.05 versus group sham, #p < 0.05
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-p
re
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-p
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-p
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Table 1 Five upregulated or downregulated miRNAs in the spinal cord of rats at 24 h after I/R injury compared with those in sham-operated rats (n = 3 per group) Fold change
P-value
rno-miR-186-5p
P-value
rno-miR-144-3 0.27492
0.0002
p
2.55371
0.0126
0.46886
0.0114
rno-miR-3568
3.57657
0.0194
rno-miR-291a-3p
rno-miR-30b-3 0.34972
0.0039
rno-miR-702-3p
p
9.81339
0.0295
3.40141
0.0005
2.6917
0.0034
rno-miR-423-5 0.48282
0.0436
rno-miR-365-3p
p
rno-miR-300-5
na ur
p
re
0.0327
lP
0.45454
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Fold change
ro of
rno-miR-129-1-3p
miRNAs
-p
miRNAs
Table 2 Representative 20 GO terms with significantly enriched function for potential targets of miR-186-5p Go Name
Gene
cell surface receptor signaling pathway
Adgrg1, Cd24, Tshr, Glp1r, Calcr, Cblb, Adgrl4, Tspan13, Mapk6, Cxcl13, Mapkapk3 Etv5, Ephb4, Rmdn3, Fgfr2, Mitf, Purb, Ets1, Nr1d2, Wnt5a, Ehf, Gli2, Elk4, Piwil4, Spast, Epha4, Etv1
cell differentiation regulation of gene expression cAMP-mediated signaling
ro of
Six1, Atxn7l3b, LOC103689943, Hey2, Mapk6, Mitf, Tob1, Sort1, Ddx3x, Tsku, Tbl1xr1, Marf1, Prkaa2 Glp1r, Lep, Ndufs4, Adcyap1, Rims2
Ank3, Dlc1, Prom2, Snx27, Gja1, Cxxc5, Cblb, Lrrc28, Depdc1b, Unc5c, Arap2, Sall1, Gabrr1, Trhde, Dcdc2, Wisp3, Ptprm, Agap1 Bnip3, Asah2, Six1, Unc5c, Casp9, Mfn2, Gja1, Rps6ka3, Rmdn3, Plagl2, Dlc1, Rnf152
apoptotic process
re
-p
signal transduction
Wnt signaling pathway, planar cell polarity pathway cell-cell signaling
Cd24, Cxcl13, Gja1, Wisp3, Bhlha15, Fgfr2, Trhde
ur
na
positive regulation of apoptotic process
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Vangl2, Wnt9b, Wnt5a
Ddx3x, Tlr3, Unc5c, Tnfsf10, Phlda3, Itga6, Camk1d, Casp9, Tomm22, Tnfrsf8, Bnip3
central nervous system
Map2, Btg2, Lep
Jo
neuron development
positive regulation of ERK1 and ERK2 cascade
Dstyk, Adcyap1, Prkca, Epha4, Sema7a, Calcr, Dnajc27, Fgfr2, Ephb4
negative regulation of
Prkca, Nf2, Chp1, Lrtm2, Lrrtm3, Ubash3b protein kinase activity
regulation of cell proliferation
Brca1, Pds5b, Fgfr2, Cxcl13, Prdm1, Tnfrsf8, Nf2, Sat1, Mitf
toll-like receptor signaling
Mapkapk3, Rps6ka3, Tlr3 pathway intrinsic apoptotic signaling
Bnip3, Ddx3x, Prkca pathway regulation of Wnt signaling
Mdfic, Vangl2, Dcdc2
ro of
pathway neuron migration
Usp9x, Gja1, Neurod4, Dcdc2, Dclk1, Dcx
neuron differentiation
Wnt9b, Wnt5a, Mtpn, Cops2, Btg2, Atp2b2, Hipk1
-p
toll-like receptor 3 signaling
Tlr3, Scara3
re
pathway regulation of inflammatory
Nr1d2, Adamts12, Brd4, Sema7a
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ur
na
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response