长链非编码 RNA NEAT1/miR-128-3p/AQP4 轴调控脊髓损伤诱导的神经性疼痛进展。
LncRNA NEAT1/miR-128-3p/AQP4 axis regulating spinal cord injury-induced neuropathic pain progression.
机构信息
Department of Anesthesiology, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China.
Department of Anesthesiology, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China.
出版信息
J Neuroimmunol. 2021 Feb 15;351:577457. doi: 10.1016/j.jneuroim.2020.577457. Epub 2020 Dec 9.
BACKGROUND
Neuropathic pain (NP) is the comorbidity in spinal cord injury(SCI), which is the hardest to cure. Non-coding RNA dysregulations are related to the development of NP. NEAT1(nuclear paraspeckle assembly transcript 1) is a new type of lncRNA. This study explores the role and specific mechanism of NEAT1 in SCI-mediated NP.
METHODS
Firstly, the NEAT1 expression in SCI rats and the control group was detected with RT-PCR to analyze the relationship between NEAT13 and NP symptoms. Then, SCI rats were intrathecally injected with NEAT13 overexpressing and knocking down lentiviruses. Afterward, ELISA was utilized to assess the expression of IL-6, IL-1β and TNFα in rats. Subsequently, immunohistochemistry was adopted to verify the activation of microglial cells. After that, bioinformatics analysis was employed to further predict the downstream target genes of NEAT1, while RT-PCR and Western blot were conducted to determine the relative expression of miR-128-3p and aquaporin-4(AQP4). Meanwhile, a dual-luciferase reporter assay was performed to further study the targeting relationship between NEAT1 and miR-128-3p, and miR-128-3p and AQP4.
RESULTS
SCI rats showed distinctly higher NEAT1 expression compared with that of the control group. ELISA experiment confirmed that the over-expression of NEAT1 enhanced the expression of IL-6, IL-1β, and TNFα in SCI rats. Other related mechanism studies revealed that NEAT13 targeted and inhibited miR-128-3p as its competing endogenous RNA (ceRNA), and enhanced AQP4 expression, while miR-128-3p targeted AQP4 to regulate its expression.
SUMMARY
NEAT1 affects AQP4 signaling pathway to alleviate the spinal cord injury-induced NP via promoting miR-128-3p expression.
背景
神经病理性疼痛（NP）是脊髓损伤（SCI）的合并症，是最难治愈的。非编码 RNA 失调与 NP 的发展有关。NEAT1（核斑组装转录本 1）是一种新型的长链非编码 RNA。本研究探讨了 NEAT1 在 SCI 介导的 NP 中的作用及其特定机制。
方法
首先，通过 RT-PCR 检测 SCI 大鼠和对照组中 NEAT1 的表达，分析 NEAT13 与 NP 症状的关系。然后，向 SCI 大鼠鞘内注射过表达和敲低的 NEAT1 慢病毒。之后，采用 ELISA 法检测大鼠 IL-6、IL-1β 和 TNFα 的表达。随后，采用免疫组织化学法验证小胶质细胞的激活情况。之后，采用生物信息学分析进一步预测 NEAT1 的下游靶基因，同时采用 RT-PCR 和 Western blot 法确定 miR-128-3p 和水通道蛋白-4（AQP4）的相对表达量。同时，进行双荧光素酶报告基因实验进一步研究 NEAT1 与 miR-128-3p 以及 miR-128-3p 与 AQP4 之间的靶向关系。
结果
SCI 大鼠的 NEAT1 表达明显高于对照组。ELISA 实验证实，过表达 NEAT1 增强了 SCI 大鼠中 IL-6、IL-1β 和 TNFα 的表达。其他相关机制研究表明，NEAT13 作为其竞争性内源 RNA（ceRNA）靶向并抑制 miR-128-3p，增强 AQP4 的表达，而 miR-128-3p 靶向 AQP4 以调节其表达。
总结
NEAT1 通过促进 miR-128-3p 的表达影响 AQP4 信号通路，从而减轻脊髓损伤引起的 NP。
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