Wei Rong, Zhang Rufang, Li Hongmei, Li Hongyun, Zhang Saiji, Xie Yewei, Shen Li, Chen Fang
Department of Anesthesiology, Shanghai Children's Hospital, Shanghai Jiao Tong University, NO. 355, Luding Road, Shanghai, 200062, China.
Department of Cardiothoracic Surgery, Shanghai Children's Hospital, Shanghai Jiao Tong University, NO. 355, Luding Road, Shanghai, 200062, China.
Curr Neurovasc Res. 2018;15(1):47-54. doi: 10.2174/1567202615666180403170902.
We previously demonstrated that microRNAs (miRNA) play an important role in Hypothermic Circulatory Arrest (DHCA)-associated neural injury. However, the specific role of miRNAs in the pathogenesis of DHCA-induced neuron death is still unclear.
Thus, in the present study, we investigated miR-29 expression and roles in neuronal HT-22 cells with Oxygen-glucose Deprivation/reoxygenation (OGD/R). In this study, the model of OGD/R was established using an airtight culture container and the anaeropack. Measurement of Reactive Oxygen Species (ROS) production and Mitochondrial Membrane Potential (MMP) was done using the probes of JC-1 and H2DCFDA. The microRNA (miRNA) profile in hippocampal neurons from rat model of DHCA was determined by miRNA deep sequencing.
We found that the expression of the miR-29 family (miR-29a/b/c) was significantly reduced in model of DHCA and OGD/R. Overexpression of the miR-29 family inhibited the OGD/R-induced elevation of ROS and reduction of MMP in HT-22 cells. In addition, administration of the miR-29 family suppressed proteins of Keap1, Bax and PUMA and increased Nrf2 expression. We further demonstrated that the miR-29 family targeted the PUMA by luciferase reporter assay and Western blot analysis.
In conclusion, our data suggest that by targeting a pro-apoptotic BCL2 family member PUMA, the miR-29 family might emerge as a strategy for protection against DHCA-mediated neural cell injury.
我们之前证明了微小RNA（miRNA）在低温循环骤停（DHCA）相关神经损伤中起重要作用。然而，miRNA在DHCA诱导的神经元死亡发病机制中的具体作用仍不清楚。
因此，在本研究中，我们研究了miR-29在氧糖剥夺/复氧（OGD/R）的神经元HT-22细胞中的表达及作用。在本研究中，使用气密培养容器和厌氧袋建立OGD/R模型。使用JC-1和H2DCFDA探针测量活性氧（ROS）生成和线粒体膜电位（MMP）。通过miRNA深度测序确定DHCA大鼠模型海马神经元中的微小RNA（miRNA）谱。
我们发现miR-29家族（miR-29a/b/c）的表达在DHCA和OGD/R模型中显著降低。miR-29家族的过表达抑制了OGD/R诱导的HT-22细胞中ROS升高和MMP降低。此外，给予miR-29家族抑制了Keap1、Bax和PUMA蛋白的表达并增加了Nrf2表达。我们通过荧光素酶报告基因测定和蛋白质印迹分析进一步证明miR-29家族靶向PUMA。
总之，我们的数据表明，通过靶向促凋亡BCL2家族成员PUMA，miR-29家族可能成为预防DHCA介导的神经细胞损伤的一种策略。
