MiR-125a-5p/132协同调节钠通道诱发学习记忆障碍的分子机制

Dementia has become a global concern of social problems. However,the pathogenesis of dementia remains unclear. Previous studies on microRNA regulation in dementia mainly paid attention to its effects on amyloidogenesis, it has not been reported on the roles of microRNA in synaptic plasticity in dementia. In this study, we meet clinical needs to investigate the molecular mechanisms of miR-125a-5p/132-mediated learning and memory impairment induced by cerebral hypoperfusion in rats by Morris water maze, patch-clamp, molecular biological techniques and so on. We also try to validate these changes in human brain tissues and from human plasma samples. Our previous results have demonstrated that the surface expression of sodium channel proteins (Nav1.1, Nav1.2, Nav1.3) was actually reduced even though the total protein levels are increased in the hippocampus of 2VO rats compared with sham rats. However,the expression of Navβ2 was significantly decreased in 2VO rats compared with sham rats. Meanwhile,miR-125a-5p was higher and miR-132 was lower in the hippocampus of 2VO rats than that in sham rats using microarrays and real-time quantitative PCR techniques. In this study, we will try to prove the roles of miR-125a-5p and miR-132 in dementia, and the mechanisms may be related to the following aspects: Firstly, miR-125a-5p/132 synergistically disturb the sodium channel protein expression(miR-132 may regulate the expression of Nav1.1, Nav1.2, Nav1.3; miR-125a-5p may regulate the expression of Navβ2 by targetscan software), suppress sodium channel trafficking to cause sodium channel dysfunction in neuron, resulting in reduced neuronal excitability. Secondly, transcription factor CREB may regulate the expression of miR-132, and miR-125a-5p may be a direct target of transcription factor E2F5. Meanwhile, miR-125a-5p has been proposed to suppress the expressions of CREB and miR-132 is supposed to target E2F5. Our study attempts to demonstrate that miR-125a-5p and miR-132 coordinatedly modulate neuronal and synaptic plasticity to reveal their new regulatory mechanisms in learning and memory impairment. This study will definitely offer the theoretical background and guidance for the prevention and treatment of dementia

老年性痴呆已成为全球关注的焦点问题，以往关于microRNA调控痴呆的研究多集中在其对Aβ生成的调控，microRNA对神经突触可塑性的调节及其在痴呆中的作用机制有待深入研究。本研究应用水迷宫、膜片钳和分子生物学等技术，探讨microRNA协同调控神经元突触可塑性诱发痴呆大鼠学习记忆障碍的新机制，并在人脑标本中进行验证。本课题前期结果显示痴呆大鼠脑组织钠通道调控紊乱，miR-125a-5p升高，miR-132下降。根据Targetscan软件预测Nav1.1, Nav1.2, Nav1.3是miR-132共同调控靶点，Navβ2是miR-125a-5p的靶点。验证miR-132和miR-125a-5p协同诱导钠通道表达、转运、功能障碍，诱发神经元兴奋性及突触传递障碍；探讨转录因子CREB及E2F5对miR-132和miR-125a-5p表达的调控作用。本研究将为临床痴呆的防治提供理论依据。

随着全球老龄化状况的加剧，老年痴呆目前已成威胁老年人健康的主要疾病，前期研究已证明双侧颈总动脉结扎引起的慢性大脑低灌注能够诱发大鼠认知记忆损伤。本项目研究结果显示大鼠在慢性大脑低灌注后海马及皮层组织中的Nav1.1和Nav1.2总蛋白的表达明显升高, Navβ2总蛋白的表达明显降低, miR-132表达显著降低, miR-125a-5p表达升高 。2VO大鼠脑立体定位注入miR-132可明显改善2VO大鼠的学习记忆能力。 在2VO模型中注射慢病毒包装的miR-132 mimics后，海马和皮层中的miR-132表达大大提升，与此同时Nav1.1和Nav1.2蛋白的表达被逆转。2VO大鼠脑立体定位注入AMO-miR-125a-5p可明显改善2VO大鼠的学习记忆能力。在2VO模型中注射miR-125a-5p inhibitor后，海马中的miR-125a-5p表达明显下降，与此同时Navβ2蛋白的表达被逆转。在离体培养的神经细胞中转入miR-132 mimics后，Nav1.1和Nav1.2表达明显下降，加入其抑制剂AMO-miR-132后，作用被逆转。转染miR-132mimics组的神经元中Navβ2表达明显下降，加入其抑制剂AMO-miR-125a-5p后，作用被逆转。荧光素酶双报告基因技术显示miR-132与SCN1A的3’UTR区有结合位点,miR-125a-5p与SCN2B的3’UTR区有结合位点。因此本项目得出以下结论：SCN1A和SCN2A是miR-132的靶基因，miR-132可通过调控钠离子通道蛋白Nav1.1和Nav1.2的表达，SCN2B是miR-125a-5p的靶基因，miR-125a-5p可通过调控钠离子通道蛋白Navβ2的表达，进而参与痴呆的发生发展，该项目为以后痴呆的临床诊断和治疗提供新的思路。

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