Long Non-coding RNA TUG1 Sponges Mir-145a-5p to Regulate Microglial Polarization After Oxygen-Glucose Deprivation
Long Non-coding RNA TUG1 Sponges Mir-145a-5p to Regulate Microglial Polarization After Oxygen-Glucose Deprivation
复制标题
长非编码 RNA TUG1 海绵 Mir-145a-5p 在缺氧和葡萄糖剥夺后调节小胶质细胞极化
DOI：
10.3389/fnmol.2019.00215
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发表时间：
2019-09-10
影响因子：
4.8
通讯作者：
Yu, Jian
中科院分区：
文献类型：
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作者：
Wang, Haoyue;Liao, Songjie;Yu, Jian
小胶质细胞通过释放多种炎性细胞因子在缺血性脑卒中后的神经炎症中起关键作用。长链非编码RNA牛磺酸上调基因1（lncRNA TUG 1）在成人脑组织中广泛表达，参与多种神经系统疾病相关的生物学过程。然而，TUG 1在小胶质细胞活化中的作用仍未确定。体外培养BV-2小胶质细胞，并使用TUG 1 siRNA敲低其RNA水平。在TUG 1 siRNA或scramble siRNA瞬时转染后，使小胶质细胞经受氧-葡萄糖剥夺（OGD）4小时。复氧24 h后，采用实时荧光定量PCR（qRT-PCR）、ELISA、免疫荧光和western blot检测SH-SY 5 Y神经母细胞瘤细胞TUG 1水平和小胶质细胞M1/M2表型，以及炎性细胞因子的释放及其对细胞活力的影响。同时，通过生物信息学分析，检测miR-145 a-5 p与TUG 1的结合情况，并通过RNA-RNA pull-down和western blot分析TUG 1与miR-145 a-5 p的相互作用以及NF-κB通路的可能参与。OGD处理后24 h，小胶质细胞中TUG 1的细胞水平瞬时上调，与下调的miR-145 a-5 p呈负相关。TUG 1基因敲除可促进小胶质细胞M1样向M2样转化，减少促炎细胞因子（肿瘤坏死因子-α，TNF-α;白细胞介素-6，IL-6）的产生，增加抗炎细胞因子（白细胞介素-10，IL-10）的释放，从而促进SH-SY 5 Y细胞的存活。同时，TUG 1基因敲低也抑制了OGD诱导的NF-κB通路的激活，表现为p-p65/p65和p-IκBα/IκBα蛋白比值的降低。此外，我们发现TUG 1可以与miR-145 a-5 p物理结合，而miR-145 a-5 p抑制剂通过激活NF-κB途径消除TUG 1敲低的保护作用，表明TUG 1与miR-145 a-5 p之间存在负相互作用。我们的研究表明，lncRNA TUG 1通过与miR-145 a-5 p的负相互作用，在OGD损伤后的相对早期阶段调节小胶质细胞极化和炎性细胞因子的产生，其中NF-κB通路可能参与其中，这可能是一个有希望的抗炎性损伤的治疗靶点。
Microglia plays a critical role in neuroinflammation after ischemic stroke by releasing diverse inflammatory cytokines. Long non-coding RNA taurine up-regulated gene 1 (lncRNA TUG1) is widely expressed in adult brain and has been reported to participate in multiple biological processes associated with nervous system diseases. However, the role of TUG1 in microglial activation remains unidentified. BV-2 microglial cells were cultured in vitro and TUG1 siRNA was used to knock down its RNA level. Microglial cells were subjected to oxygen-glucose deprivation (OGD) for 4 h following TUG1 siRNA or scramble siRNA transient transfection. After 24 h reoxygenation, TUG1 level and microglial M1/M2 phenotype, as well as releasing inflammatory cytokines and their role to viability of SH-SY5Y neuroblastoma cells were determined by quantitative real-time PCR (qRT-PCR), ELISA, immunofluorescence and western blot. In addition, miR-145a-5p, a putative microRNA to bind with TUG1 by bioinformatics analysis, was simultaneously examined, then the interaction of TUG1 with miR-145a-5p and the potential involvement of NF-κB pathway were further evaluated by RNA-RNA pull-down assay and western blot. The cellular level of TUG1 was transiently up-regulated in microglial cells 24 h after OGD treatment, with an inverse correlation to downregulated miR-145a-5p. TUG1 knockdown drove microglial M1-like to M2-like phenotypic transformation with reduced production of pro-inflammatory cytokines (tumor necrosis factor-α, TNF-α; interleukin-6, IL-6) and incremental release of anti-inflammatory cytokine (interleukin-10, IL-10), as a result, promoted the survival of SH-SY5Y cells. Meanwhile, TUG1 knockdown prevented OGD-induced activation of NF-κB pathway as well, represented by decreased ratios of p-p65/p65 and p-IκBα/IκBα proteins. Furthermore, we found that TUG1 could physically bind to miR-145a-5p while miR-145a-5p inhibitor abolished the protective effects of TUG1 knockdown through activation of NF-κB pathway, suggesting a negative interaction between TUG1 and miR-145a-5p. Our study demonstrated that lncRNA TUG1, sponging miR-145a-5p with negative interaction, could regulate microglial polarization and production of inflammatory cytokines at a relatively early stage after OGD insult, where NF-κB pathway might be involved, possibly providing a promising therapeutic target against inflammatory injury.