Xie Yun-Liang, Zhang Bo, Jing Ling
a Medical Department , The Affiliated Hospital of Bei Hua University , Jilin , China.
b Health Care Department , The Affiliated Hospital of Bei Hua University , Jilin , China.
Neurol Res. 2018 Oct;40(10):828-837. doi: 10.1080/01616412.2018.1488654. Epub 2018 Jun 29.
To explore the potential effect of miR-125b on p53-mediated regulation of Bax/Cytochrome C/Caspase-3 apoptotic signaling pathway in rats with cerebral ischemia-reperfusion (CIR) injury.
Sprague-Dawley (SD) rats were used to conduct CIR injury and injected with miR-125b mimic/inhibitor or p53 inhibitor (Pifithrin-α, PFT-α). Dual-luciferase reporter gene assay was used to analyze the targeting relationship between miR-125b and p53. Longa scoring and Triphenyl tetrazolinm chloride (TTC) staining were used to test the neurologic function and determine infarct size, respectively. Hematoxylin-eosin (HE) and Nissl's stainings were conducted to observe the morphology of cortical neurons. Neuronal nuclei (NeuN) expression was detected by immunohistochemical staining. QRT-PCR was performed to detect the expressions of miR-125b and p53. TUNEL staining and Western blotting was used to determine neuronal apoptosis and expressions of Bax/Cytochrome C/Caspase-3 signaling pathway-related proteins, respectively.
Our results showed that miR-125b could directly target p53. As observed, overexpression of miR-125b could obviously reduce the neurological score, infarct size, and brain water content after CIR in rats, which also improved the morphology of cortical neurons, increased the number of neurons, reduced neuronal apoptosis, and inhibited the expressions of Bax/Cytochrome C/Caspase-3 pathway. Moreover,the similar results were observed in rats with CIR after injected with PFT-α. But no significant differences in each index were found in CIR group and CIR + anti-miR-125b + PFT-α group.
MiR-125b exerts protective effects on CIR injury through inhibition of Bax/Cytochrome C/Caspase-3signaling pathway via targeting p53, which is likely to be a promising treatment for CIR.
3'-UTR: 3-untranslated region; CIR: cerebral ischemia-reperfusion; CIS: cerebral ischemic stroke; PFT-α: Pifithrin-α; PVDF: polyvinylidene fluoride; SD: Sprague-Dawley; TBST: tris buffered saline with tween. TTC staining: Triphenyl tetrazolinm chloride staining; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.
探讨微小RNA-125b（miR-125b）对脑缺血再灌注（CIR）损伤大鼠中p53介导的Bax/细胞色素C/半胱天冬酶-3凋亡信号通路的潜在影响。
采用斯普拉格-道利（SD）大鼠制作CIR损伤模型，并注射miR-125b模拟物/抑制剂或p53抑制剂（匹非尼酮-α，PFT-α）。采用双荧光素酶报告基因检测法分析miR-125b与p53之间的靶向关系。分别采用Longa评分和氯化三苯基四氮唑（TTC）染色检测神经功能和测定梗死灶大小。进行苏木精-伊红（HE）染色和尼氏染色以观察皮质神经元的形态。通过免疫组织化学染色检测神经元核抗原（NeuN）的表达。采用实时定量聚合酶链反应（QRT-PCR）检测miR-125b和p53的表达。分别采用末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记（TUNEL）染色和蛋白质免疫印迹法检测神经元凋亡以及Bax/细胞色素C/半胱天冬酶-3信号通路相关蛋白的表达。
结果显示，miR-125b可直接靶向p53。观察发现，miR-125b过表达可明显降低大鼠CIR后的神经功能评分、梗死灶大小和脑含水量，还可改善皮质神经元形态，增加神经元数量，减少神经元凋亡，并抑制Bax/细胞色素C/半胱天冬酶-3信号通路的表达。此外，注射PFT-α后的CIR大鼠也观察到类似结果。但CIR组与CIR +抗miR-125b + PFT-α组各指标差异无统计学意义。
miR-125b通过靶向p53抑制Bax/细胞色素C/半胱天冬酶-3信号通路，对CIR损伤发挥保护作用，这可能是一种有前景的CIR治疗方法。
3'-UTR：3'非翻译区；CIR：脑缺血再灌注；CIS：脑缺血性卒中；PFT-α：匹非尼酮-α；PVDF：聚偏二氟乙烯；SD：斯普拉格-道利；TBST：含吐温的三羟甲基氨基甲烷缓冲盐水。TTC染色：氯化三苯基四氮唑染色；TUNEL：末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记
