Nie Xiaoqi, Cheng Rui, Hao Pengfei, Guo Yuhong, Chen Gang, Ji Lei, Jia Lu
Department of Neurosurgery, Shanxi Provincial People's Hospital, No. 29, Shuangta East Street, Yingze District, Taiyuan, 030012, Shanxi, China.
Shanxi Medical University, Taiyuan, 030607, Shanxi, China.
Mol Neurobiol. 2025 Feb;62(2):2277-2291. doi: 10.1007/s12035-024-04362-7. Epub 2024 Aug 5.
This study was dedicated to investigating the effects of microRNA-128-3p (miR-128-3p) on neuronal apoptosis and neurobehavior in cerebral palsy (CP) rats via the Smurf2/YY1 axis.In vivo modeling of hypoxic-ischemic (HI) CP was established in neonatal rats. Neurobehavioral tests (geotaxis reflex, cliff avoidance reaction, and grip test) were measured after HI induction. The HI-induced neurological injury was evaluated by HE staining, Nissl staining, TUNEL staining, immunohistochemical staining, and RT-qPCR. The expression of miR-128-3p, Smurf2, and YY1 was determined by RT-qPCR and western blot techniques. Moreover, primary cortical neurons were used to establish the oxygen and glucose deprivation (OGD) model in vitro, cell viability was detected by CCK-8 assay, neuronal apoptosis was assessed by flow cytometry and western blot, and the underlying mechanism between miR-128-3p, Smurf2 and YY1 was verified by bioinformatics analysis, dual luciferase reporter assay, RIP, Co-IP, ubiquitination assay, western blot, and RT-qPCR.In vivo, miR-128-3p and YY1 expression was elevated, and Smurf2 expression was decreased in brain tissues of hypoxic-ischemic CP rats. Downregulation of miR-128-3p or overexpression of Smurf2 improved neurobehavioral performance, reduced neuronal apoptosis, and elevated Nestin and NGF expression in hypoxic-ischemic CP rats, and downregulation of Smurf2 reversed the effects of downregulation of miR-128-3p on neurobehavioral performance, neuronal apoptosis, and Nestin and NGF expression in hypoxic-ischemic CP rats, while overexpression of YY1 reversed the effects of Smurf2 on neurobehavioral performance, neuronal apoptosis, and Nestin and NGF expression in hypoxic-ischemic CP rats. In vitro, downregulation of miR-128-3p effectively promoted the neuronal survival, reduced the apoptosis rate, and decreased caspase3 protein expression after OGD, and overexpression of YY1 reversed the ameliorative effect of downregulation of miR-128-3p on OGD-induced neuronal injury. miR-128-3p targeted to suppress Smurf2 to lower YY1 ubiquitination degradation and decrease its expression.Inhibition of miR-128-3p improves neuronal apoptosis and neurobehavioral changes in hypoxic-ischemic CP rats by promoting Smurf2 to promote YY1 ubiquitination degradation and reduce YY1 expression.
本研究致力于通过Smurf2/YY1轴研究微小RNA-128-3p（miR-128-3p）对脑瘫（CP）大鼠神经元凋亡和神经行为的影响。在新生大鼠中建立缺氧缺血（HI）性CP的体内模型。在HI诱导后进行神经行为测试（趋地反射、避崖反应和抓握测试）。通过HE染色、尼氏染色、TUNEL染色、免疫组织化学染色和RT-qPCR评估HI诱导的神经损伤。通过RT-qPCR和蛋白质印迹技术测定miR-128-3p、Smurf2和YY1的表达。此外，使用原代皮质神经元在体外建立氧糖剥夺（OGD）模型，通过CCK-8法检测细胞活力，通过流式细胞术和蛋白质印迹评估神经元凋亡，并通过生物信息学分析、双荧光素酶报告基因检测、RIP、Co-IP、泛素化检测、蛋白质印迹和RT-qPCR验证miR-128-3p、Smurf2和YY1之间的潜在机制。在体内，缺氧缺血性CP大鼠脑组织中miR-128-3p和YY1表达升高，Smurf2表达降低。下调miR-128-3p或过表达Smurf2可改善缺氧缺血性CP大鼠的神经行为表现，减少神经元凋亡，并提高Nestin和NGF表达，而下调Smurf2可逆转下调miR-128-3p对缺氧缺血性CP大鼠神经行为表现、神经元凋亡以及Nestin和NGF表达的影响，而过表达YY1可逆转Smurf2对缺氧缺血性CP大鼠神经行为表现、神经元凋亡以及Nestin和NGF表达的影响。在体外，下调miR-128-3p可有效促进OGD后神经元存活，降低凋亡率，并降低caspase3蛋白表达，而过表达YY1可逆转下调miR-128-3p对OGD诱导的神经元损伤的改善作用。miR-128-3p靶向抑制Smurf2以降低YY1泛素化降解并降低其表达。抑制miR-128-3p通过促进Smurf2促进YY1泛素化降解并降低YY1表达，改善缺氧缺血性CP大鼠的神经元凋亡和神经行为变化。
