Deng Guiying, Gao Yunbing, Cen Zhongxi, He Jichen, Cao Baichuan, Zeng Gaofeng, Zong Shaohui
Research Centre for Regenerative Medicine and Guangxi Key Laboratory of Regenerative Medicine, Collaborative Innovation Center of Guangxi Biological Medicine, Guangxi Medical University, Nanning, China.
Department of Spine Osteopathia, the First Affiliated Hospital of Guangxi Medical University, Nanning, China.
Cell Physiol Biochem. 2018;50(2):512-524. doi: 10.1159/000494165. Epub 2018 Oct 11.
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.
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.
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.
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.
背景/目的：miR-136-5p通过未知机制参与脊髓损伤（SCI）后的恢复过程。我们在SCI大鼠模型中研究了miR-136-5p参与炎症反应的潜在机制。
体外培养Sprague-Dawley大鼠星形胶质细胞以构建报告质粒。采用荧光素酶测定法检测miR-136-5p靶向IKKβ和A20基因的能力。接下来，构建重组慢病毒载体，其要么过表达miR-136-5p，要么抑制其表达。在SCI大鼠模型中体内观察miR-136-5p过表达和miR-136-5p沉默对炎症的影响。检测IL-1β、IL-6、TNF-α、IFN-α及相关蛋白（A20、IKKβ和NF-κB）的表达。
体外研究表明，用miR-136-5p刺激细胞后，在IKKβ基因的3'非翻译区（UTR）存在时，荧光素酶活性显著激活。然而，在A20基因的3'UTR区存在时，荧光素酶活性显著抑制。因此，miR-136-5p可能直接作用于IKKβ和A20基因的3'UTR区以调节其表达。miR-136-5p过表达促进SCI大鼠相关细胞因子和NF-κB的产生，并抑制A20蛋白的表达。
miR-136-5p过表达促进SCI大鼠中IL-1β、IL-6、TNF-α、IFN-α、IKKβ和NF-κB的产生，但抑制A20的表达。在这些条件下，炎症细胞浸润到大鼠脊髓中增加，损伤明显加重。miR-136-5p沉默显著降低miR-136-5p过表达后所述的蛋白表达结果，并改善炎症细胞浸润和脊髓损伤。因此，miR-136-5p可能是治疗SCI的新靶点。
