MiR-27a ameliorates inflammatory damage to the blood-spinal cord barrier after spinal cord ischemia: reperfusion injury in rats by downregulating TICAM-2 of the TLR4 signaling pathway.
MiR-27a ameliorates inflammatory damage to the blood-spinal cord barrier after spinal cord ischemia: reperfusion injury in rats by downregulating TICAM-2 of the TLR4 signaling pathway.
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DOI：
10.1186/s12974-015-0246-3
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发表时间：
2015-02-07
影响因子：
9.3
通讯作者：
Ma H
中科院分区：
文献类型：
--
作者：
Li XQ;Lv HW;Wang ZL;Tan WF;Fang B;Ma H
脊髓缺血再灌注（IR）损伤引起炎症，随后增加血脊髓屏障渗漏和toll样受体4 （TLR4）通路激活。在缺血过程中，MicroRNAs （miRs）有效地调节多种靶mrna。然而，它们在IR损伤中的作用却知之甚少。我们研究了mir，特别是miR-27a，在IR后TLR4通路介导的炎症反应中的参与。我们使用基因组学方法检测大鼠在缺血14分钟后再灌注24或72小时后的miRs变化。采用定量RT-PCR技术鉴定并确认参与调控TLR4通路激活的miRs。我们扫描miR数据库寻找潜在的miR靶点，并通过定量RT-PCR确认这些靶点。从缺血前3天开始，每隔12小时在鞘内注射miR模拟物和抗miR寡核苷酸（AMOs）。通过PCR、western blotting和ELISA分析miRs对TLR4通路及下游细胞因子的影响。采用双免疫荧光染色法测定靶点与TLR4的关系。采用Evans蓝（EB）染色检测血脊髓屏障（BSCB）通透性。微阵列分析显示，在损伤后24小时，3个miRs上调（>2.0倍），15个miRs下调（<0.5倍），在72小时，与假手术对照组相比，4个miRs上调，14个miRs下调。我们将重点放在miR-27a上，预计其包含与toll样受体适配器分子2 （TICAM-2）的3'-未翻译区（UTR）互补的序列。双免疫染色显示TLR4激活与TICAM-2表达变化相关。与IR组和阴性对照组相比，鞘内输注miR-27a模拟了IR诱导的TLR4激活和BSCB的炎症损伤，在再灌注后24和72小时表现为EB外渗减少，核因子κ b （NF-κB）和白细胞介素(IL)-1β水平降低，而miR-27a AMO预处理加重了这些损伤。我们提出了第一个证据，证明mir在脊髓IR损伤中起重要作用。我们发现TICAM-2是miR-27a的新靶点。miR-27a上调通过负调控TLR4信号通路的TICAM-2和抑制NF-κB/IL-1β通路，减轻ir诱导的BSCB炎症损伤。这些结果为IR损伤的治疗提供了新的靶点。
Spinal cord ischemia reperfusion (IR) injury causes inflammation and subsequently increases blood-spinal cord barrier leakage and Toll-like receptor 4 (TLR4) pathway activation. MicroRNAs (miRs) effectively regulate numerous target mRNAs during ischemia. However, their roles during IR injury are poorly understood. We investigated miRs involvement, particularly miR-27a, in TLR4 pathway-mediated inflammatory responses after IR. We used a genomics approach to examine changed miRs of rats that had undergone 14 minutes of ischemia, followed by 24 or 72 hours of reperfusion. Quantitative RT-PCR was used to identify and confirm the miRs involved in regulating TLR4 pathway activation. We scanned miR databases for potential miR targets and confirmed these targets by quantitative RT-PCR. The miR mimic and anti-miR oligonucleotides (AMOs) were intrathecally injected at 12-hour intervals beginning three days before the ischemia. The effects of miRs on the TLR4 pathway and downstream cytokines were analyzed by PCR, western blotting, and ELISA. Double immunofluorescence staining was perfumed to determine the relationship between the targets and TLR4. Blood-spinal cord barrier (BSCB) permeability was examined using Evans blue (EB) dye. A microarray analysis revealed that at 24 hours post-injury, three miRs were upregulated (>2.0 fold) and 15 miRs were downregulated (<0.5 fold), and at 72 hours, four miRs were upregulated and 14 were downregulated compared to their levels in sham-operated controls. We focused on miR-27a, which is predicted to contain sequences complementary to the 3'-untranslated region (UTR) of Toll-like receptor adaptor molecule 2 (TICAM-2). Double immunostaining indicated that TLR4 activation correlated with changes in TICAM-2 expression. Compared to the rats in the IR and negative control groups, intrathecal infusion of the miR-27a mimic attenuated IR-induced TLR4 activation and inflammatory damage to the BSCB, which was shown as decreased EB extravasation and lower levels of nuclear factor kappa-B (NF-κB) and lnterleukin (IL)-1β at 24 and 72 hours after reperfusion, whereas pretreatment with miR-27a AMO aggravated these injuries. We present the first evidence that miRs play an important role in spinal cord IR injury. We identified TICAM-2 as a novel target of miR-27a. miR-27a upregulation attenuates IR-induced inflammatory damage to the BSCB by negatively regulating TICAM-2 of the TLR4 signaling pathway and inhibiting the NF-κB/IL-1β pathway. These results provide new therapeutic targets for IR injury treatment.
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影响因子：
9.3
作者：
Li XQ;Lv HW;Tan WF;Fang B;Wang H;Ma H
通讯作者：
Ma H
影响因子：
3.7
作者：
Liu G;Cao P;Chen H;Yuan W;Wang J;Tang X
通讯作者：
Tang X
影响因子：
4.3
作者：
Fan, Lihong;Wang, Kunzheng;Dang, Xiaoqian
通讯作者：
Dang, Xiaoqian
影响因子：
38.1
作者：
Bhalala, Oneil G.;Srikanth, Maya;Kessler, John A.
通讯作者：
Kessler, John A.
影响因子：
9.3
作者：
Hua F;Wang J;Ishrat T;Wei W;Atif F;Sayeed I;Stein DG
通讯作者：
Stein DG