MicroRNA-424靶向NFI-A调控不同年龄小鼠脑缺血后星形胶质细胞分化的神经重塑机制

After cerebral ischemia, astrocyte proliferation lessens recovery of neurological function due to glial scar formation, which inhibits neurons regeneration. Our previous study found that miR-424 is neuroprotective in the acute phase of cerebral ischemia, and targeted inhibition of the NFI-A expression in neural cells. NFI-A/DNMT1 mediated methylation and epigenetic regulation of astrocytes-specific gene expression is an important target for neural stem cells differentiation into astrocytes. That reveals that miR-424 targeting NFI-A pathway may play an important role in differentiation of neural cells in the repair period after cerebral ischemia. Therefore, this study using laser capture, methylation PCR, chromatin immunoprecipitation, viral infections and other technologies, elucidates the effects of miR-424 on differentiation of astrocytes proliferation and methylation regulation mechanism in the chronic phase after cerebral ischemia, demonstrates the important role of miR-424 in the repair period. MiR-424 targeted inhibition of transcription factor NFI-A expression in neural precursor cells, the DNA methyltransferase DNMT1 was increased, and then make the astrocyte specific gene GFAP promoter methylation of DNA increased, then the binding of NFI-A and STAT3 decreased, thereby reducing the astrocyte specific gene expression. Our investigation will provide new therapeutic targets and the corresponding theoretical and experimental basis to regulation of differentiation of neural stem cells into astrocytes, neurons, oligodendrocytes in recovery period after cerebral ischemia.

脑缺血后，星形胶质细胞活化增殖形成胶质瘢痕，抑制神经元发生、限制神经功能恢复。转录因子NFI-A抑制DNMT1催化的GFAP启动子区DNA甲基化，是诱导神经前体细胞向星形胶质细胞分化的关键机制。我们研究发现miR-424对脑缺血具有重要的神经保护作用，且靶向NFI-A，提示miR-424可能在脑缺血神经前体细胞分化中发挥重要作用。本研究采用不同年龄小鼠模型，结合病毒感染、激光捕获、甲基化测序、ChIP-PCR、RISC免疫共沉淀方法，验证假说：miR-424靶向神经前体细胞中NFI-A，增加DNMT1活性，提高星形胶质细胞特异基因的启动子区DNA甲基化，抑制转录因子NFI-A和STAT3的结合，从而降低星形胶质细胞特异基因表达，减少胶质瘢痕形成，促进神经元和少突胶质细胞生成。本项目通过阐明miR-424促进脑缺血神经重塑作用及关键机制，为不同年龄脑卒中患者神经重塑的治疗提供新思路、新靶标。

脑缺血后星形胶质细胞的DNA和组蛋白甲基化模式受年龄的影响，但在老年啮齿动物脑缺血卒中后，异常甲基化是否能诱发反应性星形胶质细胞增生尚不清楚。在9月龄大脑中动脉闭塞（MCAO）雄性小鼠脑缺血再灌注后1、3、14天，磷酸化信号转导子和转录激活因子3（STAT3）水平随星形胶质细胞增生标志物胶质纤维酸性蛋白（GFAP）水平升高而升高。再灌注后3天和14天，GFAP基因启动子中STAT3结合位点的甲基化增加。抑制性修饰组蛋白3-赖氨酸27-三甲基化（H3K27me3）降低，而促进转录的修饰组蛋白3-赖氨酸4-三甲基化（H3K4me3）增加。缺血脑星形胶质细胞DNA甲基转移酶1（DNMT1）表达上调。在原代星形胶质细胞培养中，miR-424以核因子IA（NFIA）为靶点；miR-424 agomir在缺氧缺糖和诱导原代星形胶质细胞周期阻滞的同时，提高了U87细胞DNMT1和H3K27me3的水平，从而保护MCAO小鼠神经元及其轴突的结构。这些结果表明miR-424通过NFIA/DNMT1信号增强H3K27me3对老年小鼠脑缺血再灌注后星形胶质细胞增生具有预防作用。

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