miR-136-5p Regulates the Inflammatory Response by Targeting the IKK/NF-B/A20 Pathway After Spinal Cord Injury
miR-136-5p Regulates the Inflammatory Response by Targeting the IKK/NF-B/A20 Pathway After Spinal Cord Injury
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miR-136-5p 通过靶向脊髓损伤后的 IKKβ/NF-κB/A20 通路来调节炎症反应
DOI：
10.1159/000494165
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发表时间：
2018-01-01
影响因子：
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通讯作者：
Zong, Shaohui
中科院分区：
文献类型：
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作者：
Deng, Guiying;Gao, Yunbing;Zong, Shaohui
背景/目的：miR-136-5p通过一种未知的机制参与脊髓损伤(SCI)后的康复。我们研究了miR-136-5p参与脊髓损伤大鼠炎症反应的机制。方法：体外培养SD大鼠星形胶质细胞，构建报告基因表达载体。荧光素酶分析检测miR-136-5p靶向IKK、β和A20基因的能力。其次，构建了miR-136-5p过表达或抑制其表达的重组慢病毒载体。在大鼠脊髓损伤模型中，观察miR-136-5p过表达和miR-136-5p沉默对炎症反应的影响。检测IL-1β、IL-6、肿瘤坏死因子-α、干扰素-α及相关蛋白(A20、IKKβ、NF-κB)的表达。结果：体外实验表明，经miR-136-5p刺激后，IKKβ基因3‘端非翻译区的荧光素酶活性明显激活。然而，在A20基因的3‘非编码区的存在下，荧光素酶的活性被显著抑制。因此，miR-136-5p可能直接作用于IKKβ和A20基因的3‘非编码区，调节其表达。MIR-136-5p过表达促进脊髓损伤大鼠相关细胞因子和NF-κB的产生，抑制A20蛋白的表达。结论：miR-136-5p过表达可促进脊髓损伤大鼠IL-1β、IL-6、肿瘤坏死因子-α、干扰素-α、IKKβ和核因子-κB的产生，但抑制A20的表达。在这种情况下，炎性细胞在大鼠脊髓内的浸润增加，损伤明显加重。MiR-136-5p的沉默显著降低了miR-136-5p过表达后的蛋白表达结果，并改善了炎症细胞的浸润和对脊髓的损伤。因此，miR-136-5p可能成为治疗脊髓损伤的新靶点。
Background/Aims: miR-136-5p participates in recovery after spinal cord injury (SCI) via an unknown mechanism. We investigated the mechanism underlying the involvement of miR-136-5p in the inflammatory response in a rat model of SCI. Methods: Sprague-Dawley rat astrocytes were cultured in vitro to construct a reporter plasmid. Luciferase assays were used to detect the ability of miR-136-5p to target the IKKβ and A20 genes. Next, recombinant lentiviral vectors were constructed, which either overexpressed miR-136-5p or inhibited its expression. The influence of miR-136-5p overexpression and miR-136-5p silencing on inflammation was observed in vivo in an SCI rat model. The expression of IL-1β, IL-6, TNF-α, IFN-α, and related proteins (A20, IKKβ, and NF-κB) was detected. Results: In vitro studies showed that luciferase activity was significantly activated in the presence of the 3’ untranslated region (UTR) region of the IKKβ gene after stimulation of cells with miR-136-5p. However, luciferase activity was significantly inhibited in the presence of the 3’UTR region of the A20 gene. Thus, miR-136-5p may act directly on the 3’UTR regions of the IKKβ and A20 genes to regulate their expression. miR-136-5p overexpression promoted the production of related cytokines and NF-κB in SCI rats and inhibited the expression of A20 protein. Conclusion: Overexpression of miR-136-5p promotes the generation of IL-1β, IL-6, TNF-α, IFN-α, IKKβ, and NF-κB in SCI rats but inhibits the expression of A20. Under these conditions, inflammatory cell infiltration into the rat spinal cord increases and injury is significantly aggravated. Silencing of miR-136-5p significantly reduces the protein expression results described after miR-136-5p overexpression and ameliorates the inflammatory cell infiltration and damage to the spinal cord. Therefore, miR-136-5p might be a new target for the treatment of SCI.