Purpose

Cerebral ischemic injury contributes to severe dysfunction of the brain, which triggers extremely high mortality and disability. The role of microRNA (miR)-181a-5p is documented in cerebral ischemic injury. Therefore, this study intended to further figure out the mechanism of miR-181a-5p in cerebral ischemic injury.

Methods

miR-181a-5p expression in middle cerebral artery occlusion (MCAO) mouse model, oxygen-glucose-deprivation/reoxygenation (OGD/R) N2a cell model, and serum from acute ischemic injury (ACI) patients was evaluated using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Gain- and loss-of-function assays were implemented in MCAO mice and OGD/R-induced N2a cells. In mice, the cerebral infarction area was assessed with 2,3,5-triphenyltetrazolium chloride staining, the number of damaged neurons by Nissl staining, and apoptosis by TdT-mediated dUTP-biotin nick end-labeling staining. Moreover, N2a cell apoptosis and proliferation were determined with flow cytometry or 5-ethynyl-2’-deoxyuridine staining, respectively. The expression of En2 and Wnt/β-catenin pathway-related factors was determined with RT-qPCR and Western blot analysis. The targeting relationship between miR-181a-5p and En2 was evaluated by dual luciferase reporter gene assay.

Results

miR-181a-5p was highly expressed in serum of ACI patients, MCAO mice, and OGD/R-induced N2a cells. En2, lowly expressed in MCAO mice, was targeted by miR-181a-5p, and miR-181a-5p down-regulation activated the Wnt/β-catenin pathway. Furthermore, miR-181a-5p inhibition or En2 overexpression reduced cerebral infarction area, the number of damaged neurons, and apoptosis in MCAO mice, and also diminished apoptosis and accelerated proliferation of OGD/R-induced N2a cells.

Conclusion

miR-181a-5p suppression activated Wnt/β-catenin pathway and sequentially attenuated cerebral ischemic injury by targeting En2.

中文翻译：

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下调miR-181a-5p可通过上调En2和激活Wnt /β-catenin途径来预防脑缺血性损伤

目的

脑缺血性损伤会导致严重的大脑功能障碍，从而引发极高的死亡率和残疾。microRNA（miR）-181a-5p在脑缺血性损伤中有作用。因此，本研究旨在进一步阐明miR-181a-5p在脑缺血性损伤中的作用机制。

方法

使用逆转录定量聚合酶评估了大脑中动脉闭塞（MCAO）小鼠模型，氧葡萄糖剥夺/复氧（OGD / R）N2a细胞模型和急性缺血性损伤（ACI）患者血清中miR-181a-5p的表达连锁反应（RT-qPCR）。在MCAO小鼠和OGD / R诱导的N2a细胞中进行了功能获得和功能丧失测定。在小鼠中，用2,3,5-三苯基四唑氯化物染色，尼氏染色对受损神经元的数量以及TdT介导的dUTP-生物素缺口末端标记染色对凋亡的评估。此外，分别通过流式细胞术或5-乙炔基-2'-脱氧尿苷染色确定N2a细胞凋亡和增殖。通过RT-qPCR和Western blot分析确定En2和Wnt /β-catenin途径相关因子的表达。

结果

miR-181a-5p在ACI患者，MCAO小鼠和OGD / R诱导的N2a细胞的血清中高度表达。En2在MCAO小鼠中低表达，被miR-181a-5p靶向，而miR-181a-5p下调激活了Wnt /β-catenin途径。此外，miR-181a-5p抑制或En2过表达减少了MCAO小鼠的脑梗死面积，受损的神经元数量和细胞凋亡，并且还减少了OGD / R诱导的N2a细胞的凋亡和加速增殖。

结论

通过靶向En2，miR-181a-5p抑制可激活Wnt /β-catenin途径，从而减轻脑缺血损伤。