Down‐regulation of miR‐3068‐3p enhances kcnip4‐regulated A‐type potassium current to protect against glutamate‐induced excitotoxicity
Down‐regulation of miR‐3068‐3p enhances kcnip4‐regulated A‐type potassium current to protect against glutamate‐induced excitotoxicity
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DOI：
10.1111/jnc.14932
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发表时间：
2019-12
影响因子：
4.7
通讯作者：
Zi-jun Su;Xu‐Yi Wang;Chen Zhou;Zhen Chai
中科院分区：
文献类型：
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作者：
Zi-jun Su;Xu‐Yi Wang;Chen Zhou;Zhen Chai
缺血性脑卒中中兴奋性毒性神经元死亡的主要原因是谷氨酸的大量释放。最近，人们发现microRNAs （miRNAs）在脑卒中病理中起着重要作用，尽管其分子机制仍有待研究。在这里，为了确定潜在的候选miRNA参与兴奋性毒性，我们用谷氨酸处理大鼠初级皮质神经元，发现miR - 3068 - 3p，一种新的miRNA，上调。我们假设恢复miR - 3068 - 3p的表达可能会影响神经元损伤的结果。使用强诱饵慢病毒抑制miR - 3068 - 3p，显著减弱了谷氨酸对神经元活力和细胞内钙超载的影响。为了揭示其机制，我们采用生物信息学分析和RNA测序来鉴定下游靶基因。另外的荧光素酶测定和western blots证实kcnip4是Kv4介导的a型钾电流（IA）调节剂，是miR‐3068‐3p的直接靶点。miR - 3068 - 3p的抑制增加了kcnip4的表达，反之亦然。此外，shRNA敲低kcnip4可消除miR - 3068 - 3p的保护作用，仅过表达kcnip4就足以在兴奋性毒性中发挥神经保护作用。此外，miR‐3068‐3p的抑制增强了IA的密度，IA的药理学抑制消除了miR‐3068‐3p抑制和kcnip4过表达的保护作用。因此，我们得出结论，miR - 3068 - 3p的抑制通过其靶基因kcnip4和kcnip4调控的IA来保护兴奋性毒性。我们的数据表明miR - 3068 - 3p/kcnip4轴可能作为缺血性卒中治疗的新靶点。
The main cause of excitotoxic neuronal death in ischemic stroke is the massive release of glutamate. Recently, microRNAs (miRNAs) have been found to play an essential role in stroke pathology, although the molecular mechanisms remain to be investigated. Here, to identify potential candidate miRNAs involved in excitotoxicity, we treated rat primary cortical neurons with glutamate and found that miR‐3068‐3p, a novel miRNA, was up‐regulated. We hypothesized that restoring miR‐3068‐3p expression might influence the neuronal injury outcomes. The inhibition of miR‐3068‐3p, using tough decoy lentiviruses, significantly attenuated the effects of glutamate on neuronal viability and intracellular calcium overload. To unravel the mechanisms, we employed bioinformatics analysis and RNA sequencing to identify downstream target genes. Additional luciferase assays and western blots validated kcnip4, a Kv4‐mediated A‐type potassium current (IA) regulator, as a direct target of miR‐3068‐3p. The inhibition of miR‐3068‐3p increased kcnip4 expression and vice versa. In addition, the knockdown of kcnip4 by shRNA abolished the protective effect of miR‐3068‐3p, and over‐expressing kcnip4 alone was sufficient to play a neuroprotective role in excitotoxicity. Moreover the inhibition of miR‐3068‐3p enhanced the IA density, and the pharmacological inhibition of IA abrogated the protective role of miR‐3068‐3p inhibition and kcnip4 over‐expression. Therefore, we conclude that inhibition of miR‐3068‐3p protects against excitotoxicity via its target gene, kcnip4, and kcnip4‐regulated IA. Our data suggest that the miR‐3068‐3p/kcnip4 axis may serve as a novel target for the treatment of ischemic stroke.