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. 2020 Mar 27;40(3):BSR20193315.
doi: 10.1042/BSR20193315.

MicroRNA-129-5p Alleviates Spinal Cord Injury in Mice via Suppressing the Apoptosis and Inflammatory Response Through HMGB1/TLR4/NF-κB Pathway

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Free PMC article

MicroRNA-129-5p Alleviates Spinal Cord Injury in Mice via Suppressing the Apoptosis and Inflammatory Response Through HMGB1/TLR4/NF-κB Pathway

Guang Wan et al. Biosci Rep. .
Free PMC article

Abstract

Secondary injury after spinal cord injury (SCI) is one reversible pathological change mainly involving excessive inflammatory response and neuro-apoptosis. Since in recent years, microRNAs (miRNAs) have been proposed as novel regulators of inflammation in different disease conditions. However, the role of miRNAs in the inflammatory response and apoptosis of secondary injury after SCI remains to be fully elucidated. Here, we tried to explore the influence and mechanism of miRNAs on the neuron inflammatory response and apoptosis after SCI. The expression profiles of miRNA were examined using miRNA microarray, and among the candidate miRNAs, miR-129-5p was found to be the most down-regulated miRNA in spinal tissues. Overexpression of miR-129-5p using agomir-miR-129-5p promoted injury mice functional recovery, suppressed the apoptosis and alleviated inflammatory response in spinal tissues. Using LPS-induced BV-2 cell model, we found miR-129-5p was also proved in protecting inflammatory response and cell apoptosis in vitro. High-mobility group protein B1 (HMGB1), a well-known inflammatory mediator, was found to be directly targeted by miR-129-5p and it was associated with the inhibitory effect of miR-129-5p on the activation of toll-like receptor (TLR)-4 (TLR4)/ nuclear factor-κB (NF-κB) pathway in vitro and in vivo. Further experiments revealed that the anti-apoptosis and anti-inflammatory effects of miR-129-5p were reversed by HMGB1 overexpression in BV-2 cells. Collectively, these data revealed that miR-129-5p alleviated SCI in mice via suppressing the apoptosis and inflammatory response through HMGB1//TLR4/NF-κB pathway. Our data suggest that up-regulation of miR-129-5p may be a novel therapeutic target for SCI.

Keywords: HMGB1//TLR4/NF-κB pathway; Spinal cord injury; inflammatory response; miR-129-5p.

Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. miR-129-5p was down-regulated in spinal cord of SCI rats
(A) The BBB scores at 1, 3, 7, 14, 21, and 28 days after SCI were shown for all groups of mice. (B) Cresyl Violet staining was used to assess spared tissue following behavioral analyses at 28 days post-injury. (C) Spinal cord water content was assessed using wet-to-dry weight method. (D) Heatmap of normalized expression levels of miRNAs in spinal cord tissues from SCI and sham rats. (E) qRT-PCR was performed to determine the expression levels of miR-129-5p in spinal cord tissues from mice at 1, 7, 14, 21, and 28 days after SCI. Data represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01 vs. Sham group.
Figure 2
Figure 2. Agomir-miR-129-5p improves recovery of SCI mice by reducing apoptosis
The mice were subjected to SCI and then treated with agomir-miR-129-5p/agomir-NC (50 μl/day, 100 nmol/ml) for 3 days (0, 1, and 2 days) via intrathecal injection starting 15 min after contusion SCI. At indicated time, the animals were killed and subsequently, a 10-mm long segment of the spinal cord was harvested for further experiments. (A) qRT-PCR was performed to determine the expression levels of miR-129-5p in spinal cord tissues at 1, 3, 7, 14, and 28 days after agomir-129-5p injection. (B) The BBB scores at 1, 3, 7, 14, 21, and 28 days after SCI were shown for all groups of mice. (C) Cresyl Violet staining was used to assess spared tissue following behavioral analyses at 28 days post-injury. (D) Spinal cord water content was assessed using wet-to-dry weight method. (E) IHC analysis of cleaved caspase-3 at 7 days post-injury in spinal cord tissues. (F) The protein expressions of Bcl-2, Bax, cleaved caspase-3 and cleaved PARP were measured by Western blot. Data represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01 vs. Sham group; ##P<0.01 vs. SCI + agomir-NC group.
Figure 3
Figure 3. miR-129-5p inhibited the inflammatory response in SCI mice
The mice were subjected to SCI and then treated with agomir-miR-129-5p/agomir-NC (50 μl/day, 100 nmol/ml) for 3 days (0, 1 and 2 days) via intrathecal injection starting 15 min after SCI. At indicated time, the animals were killed, and subsequently serum samples were collected for detection of production of inflammatory factors. The levels of TNF-α (A), IL-6 (B), IL-1β (C) and IL-10 (D) in serum of mice were measured by ELISA kits. Data represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01 vs. Sham group; ##P<0.01 vs. SCI + agomir-NC group.
Figure 4
Figure 4. Overexpression of miR-129-5p suppressed inflammatory response and apoptosis in SCI cell model
(A) BV-2 cells were treated with different concentrations of LPS (10, 100, and 1000 ng/ml) for 24 h, and the expression of miR-129-5p was detected by qRT-PCR analysis. (B) Agomir-miR-129-5p was added to the cultured BV-2 cells 4 h prior to LPS treatment and incubated for 24 h, and then the transfected efficiency of agomir-miR-129-5p was detected by qRT-PCR analysis. (C) Activity of caspase-3 was measured using a caspase-3 activity assay kit. (D) The protein expression level of caspase-3 in BV-2 cells was detected by IFA. (EH) The expressions of TNF-α, IL-6, IL-1β, and IL-10, were measured by ELISA analysis. Data represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01 vs. Control group; ##P<0.01 vs. LPS + agomir-NC group.
Figure 5
Figure 5. HMGB1 is a direct target of miR-129-5p in BV-2 cells
(A) Putative binding site of miR-129-5p and HMGB1 with mut and wt 3′UTRs. (B) Luciferase assay of BV-2 cells co-transfected with firefly luciferase constructs containing the HMGB1 wild-type or mutated 3′-UTRs and agomir-miR-129-5p or agomir-NC, as indicated (n=3). Data represent the mean ± SD of three independent experiments. **P<0.01 vs. agomir-NC group. (C) The protein levels of HMGB1 were detected by Western blot after agomir-miR-129-5p transfection. (D) BV-2 cells were treated with different concentrations of LPS (10, 100 and 1000 ng/ml) for 24 h, and the expression of HMGB1 was detected by qRT-PCR analysis. (E) The protein expression levels of HMGB1, TLR4, p-p65 and total p65 were detected by Western blot analysis. Data represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01 vs. Control group; ##P<0.01 vs. LPS + agomir-NC group.
Figure 6
Figure 6. miR-129-5p protects BV-2 cells from LPS-induced apoptosis and inflammatory response by targeting HMGB1
Agomir-miR-129-5p and pcDNA-HMGB1 were co-transfected into the cultured BV-2 cells 4 h prior to LPS treatment, and incubated for 24 h, then cells were harvested for next experiments. (A) The transfected efficiency of pcDNA-HMGB1 was determined by Western blot. (B) The protein expression level of caspase-3 was detected by IFA in BV-2 cells. (C) Activity of caspase-3 was measured using a commercial kit. (DG) The expressions of TNF-α, IL-6, IL-1β, and IL-10 were measured by ELISA analysis. Data represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01 vs. LPS group; ##P<0.01 vs. LPS + agomir-miR-129-5p group.

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