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。在新生大鼠中建立了缺氧缺血(HI)CP的体内模型。神经行为测试(地轴反射,悬崖回避反应,和抓地力测试)是在HI诱导后测量的。HE染色评价HI诱导的神经损伤,尼氏染色,TUNEL染色,免疫组织化学染色,和RT-qPCR。miR-128-3p的表达,通过RT-qPCR和蛋白质印迹技术测定Smurf2和YY1。此外,原代皮层神经元建立体外氧糖剥夺(OGD)模型,通过CCK-8测定法检测细胞活力,通过流式细胞术和蛋白质印迹评估神经元凋亡,以及miR-128-3p之间的潜在机制,通过生物信息学分析验证了Smurf2和YY1,双荧光素酶报告分析,RIP,共同IP,泛素化测定,westernblot,和RT-qPCR。在体内,miR-128-3p和YY1表达升高,缺氧缺血性CP大鼠脑组织中Smurf2表达降低。miR-128-3p的下调或Smurf2的过表达改善了神经行为表现,减少神经元凋亡,缺氧缺血性CP大鼠Nestin和NGF表达升高,Smurf2的下调逆转了miR-128-3p下调对神经行为表现的影响,神经元凋亡,Nestin和NGF在缺氧缺血性CP大鼠中的表达,而YY1的过表达逆转了Smurf2对神经行为表现的影响,神经元凋亡,Nestin和NGF在缺氧缺血性CP大鼠中的表达。体外,miR-128-3p下调可有效促进神经元存活,降低细胞凋亡率,OGD后caspase3蛋白表达降低,YY1的过表达逆转了miR-128-3p下调对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,降低YY1表达,改善缺氧缺血性CP大鼠神经元凋亡和神经行为学改变。
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.