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, 11 (6), 2948-2958
eCollection

microRNA-182-5p Alleviates Spinal Cord Injury by Inhibiting Inflammation and Apoptosis Through Modulating the TLR4/NF-κB Pathway

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microRNA-182-5p Alleviates Spinal Cord Injury by Inhibiting Inflammation and Apoptosis Through Modulating the TLR4/NF-κB Pathway

Junfeng Zhang et al. Int J Clin Exp Pathol.

Abstract

Inflammatory response and apoptosis play an important role in progression of spinal cord injury (SCI). Recently, aberrant microRNAs (miRNAs) have emerged as a key regulator in SCI. However, it remains unknown whether and how miRNAs mediated the inflammatory response after SCI. The aim of this study was to evaluate the potential role of miRNAs in SCI and elucidate underlying molecular mechanisms. First, we analyzed the microRNA expression profile in spinal cords from rats following SCI, using miRNA microarray. Interestingly, miR-182-5p was one of miRNAs most significantly downregulated in SCI. It has been reported as an inflammation suppressor in different organ injury models. Here, we used a cell model to verify the regulatory function and mechanism of miR-182-5p on inflammatory response in SCI. Overexpression of miR-182-5p attenuated H2O2-induced inflammation as reflected by reduction in proinflammatory cytokines in C8-D1A cells. Meanwhile, enhanced miR-182-5p expression significantly suppressed H2O2-induced apoptosis. Toll-like receptor 4 (TLR4), an important regulator of nuclear factor kappa-B (NF-κB) signaling pathway, was identified as a novel target of miR-182-5p in C8-D1A cells. Furthermore, overexpression of TLR4 reversed inhibitory effects of miR-182-5p overexpression on inflammation and apoptosis. More importantly, we found that miR-182-5p blocked phosphorylation of nuclear p65 and promoted phosphorylation of IκB-α in H2O2-treated C8-D1A cells. Our results confirm that miR-182-5p alleviates inflammation and apoptosis via inactivation of TLR4/NF-κB pathway in an H2O2-induced cell model. Our findings suggest that miR-182-5p may be a potential therapeutic target of SCI in the future.

Keywords: Spinal cord injury; TLR4/NF-κB pathway; apoptosis; inflammation; microRNA-182-5p.

Conflict of interest statement

None.

Figures

Figure 1
Figure 1
miR-182-5p is downregulated in rats after spinal cord injury (SCI). A. Heat map of miRNA profiles represented the significantly regulated miRNAs. The color code in the heat maps is linear with green as the lowest and red as the highest. miRNAs that were upregulated are shown in green to red, whereas miRNAs that were downregulated are shown from red to green. B. miR-182-5p expression was validated by qRT-PCR in rats after SCI (n = 6). P < 0.01 vs. Sham group. C. Expression of p-p65 in SCI rats was measured by Western Blot. (n = 2). Data represents the mean ± SD of three independent experiments. **P < 0.01 vs. Sham group.
Figure 2
Figure 2
Overexpression of miR-182-5p suppressed apoptosis in H2O2 treated C8-D1A cells. A. Expression level of miR-182-5p in C8-D1A cells was elevated by transfection of miR-182-5p mimics in H2O2 treated C8-D1A cells as measured by qRT-PCR. Data represents the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. untreated group. B. Transfection of miR-182-5p mimics significantly increased expression level of miR-182-5p in C8-D1A cells. Data represent the mean ± SD of three independent experiments. **P < 0.01 vs. control group. C. Apoptosis was detected by flow cytometry in H2O2 treated C8-D1A cells after transfection with miR-182-5p mimics. Data represent the mean ± SD of three independent experiments. **P < 0.01 vs. control group. ##P < 0.01 vs. H2O2 alone group. D. Protein expression levels of cleaved-caspase 3 and total caspase 3 were detected by Western blot in H2O2 treated C8-D1A cells after transfection with miR-182-5p mimics.
Figure 3
Figure 3
Overexpression of miR-182-5p attenuated H2O2-induced inflammation response. C8-D1A cells were transfected with miR-182-5p mimics for 48 hours and then exposed to 200 μM H2O2 for 10 h. TNF-α (A), IL-6 (B), and IL-1β (C) levels were measured by ELISA assay. Data represents the mean ± SD of three independent experiments. **P < 0.01 vs. control group. ##P < 0.01 vs. H2O2 alone group.
Figure 4
Figure 4
TLR4 is a direct target of miR-182-5p. A, B. The putative binding site of miR-182-5p and TLR4 is shown. C. Luciferase assay of HEK293 cells co-transfected with firefly luciferase constructs containing the TLR4 wild-type or mutated 3’-UTRs and miR-182-5p mimics, mimics NC, miR-182-5p inhibitor or inhibitor NC, as indicated (n = 3). Data represents the mean ± SD of three independent experiments. **P < 0.01 vs. mimics NC, ##P < 0.01 vs. inhibitor NC. D, E. Expression of TLR4 mRNA and protein after transfection with miR-182-5p mimic or miR-182-5p inhibitor was measured by qRT-PCR and Western blot. Data represents the mean ± SD of three independent experiments. **P < 0.01 vs. inhibitor NC, ##P < 0.01 vs. mimics NC. F. C8-D1A cells were transfected with miR-182-5p mimics or miR-182-5p inhibitor for 48 hours and then exposed to 200 μM H2O2 for 10 h. TLR4 expression was assessed by qRT-PCR. Data represents the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. control group. ##P < 0.01 vs. H2O2 + mimics NC group. &&P < 0.01 vs. H2O2 + inhibitor NC group.
Figure 5
Figure 5
TLR4 overexpression abrogates the inhibitory effects of miR-182-5p mimics on H2O2 treated C8-D1A cells. C8-D1A cells were co-transfected with miR-182-5p mimics and pcDNA-TLR4 for 48 h and then exposed to 200 μM H2O2 for 10 h. A. Apoptosis was detected by flow cytometry. B-D. TNF-α, IL-6, and IL-1β levels in were measured by ELISA assay. Data represents the mean ± SD of three independent experiments. **P < 0.01 vs. H2O2 alone group, ##P < 0.01 vs. H2O2 + miR-182-5p mimics group.
Figure 6
Figure 6
Overexpression of miR-182-5p blocked TLR4/NF-κB pathway in SCI cell model. C8-D1A cells were co-transfected with miR-182-5p mimics and pcDNA-TLR4 vector for 48 hours and then exposed to 200 μM H2O2 for 10 h. A. The levels of TLR4, nuclear p-p65, and p-IκB-α were measured by Western blot. B. The bands were semi-quantitatively analyzed by using Image J software, normalized to β-actin density. Data represents the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. H2O2 alone group. ##P < 0.01 vs. H2O2 + miR-182-5p mimics group.

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