miR-130a通过调控PTEN对缺血性脑卒中神经功能的影响

Effects of mir-130a on neurological function in ischemic stroke by regulating PTEN

目的探究miR-130a通过调控磷酸酶张力蛋白同源物基因(PTEN)对缺血性脑卒中大鼠神经功能恢复的机制。方法60只雄性SD大鼠随机分为对照组(假手术处理,n=20)、模型组(手术建立缺血性脑卒中模型,n=20)和模拟转染组(手术建立缺血性脑卒中模型后,将miR-130a模拟转染注射到大鼠的侧脑室,n=20),通过神经损伤严重程度评分量表(mNSS)评估手术后第1、10和25天时的神经缺陷程度;用RT-PCR分析用miR-130a和PTEN表达;荧光素酶法测定野生型-PTEN和突变型-PTEN表达的荧光素酶活性,酶联免疫吸附实验检测各组大鼠脑组织白介素-6(IL-6)、白介素-10(IL-10)及肿瘤坏死因子-α(TNF-α)的含量;TUNEL染色检测脑组织神经元凋亡水平,蛋白印迹法分析磷酸化蛋白激酶B(p-AKT)、磷酸化糖原合成酶激酶-3β(p-GSK3β)及磷酸化原癌基因丝苏氨酸蛋白激酶(p-c-Raf)表达。结果miR-130a模型组大鼠的mNSS评分,miR-130a的表达减轻神经细胞的功能损伤;与对照组比较,模型组的miR-130a mRNA、p-AKT、p-GSK3β及p-c-Raf表达降低,TUNEL阳性细胞数量和PTEN mRNA表达升高(P<0.05);与模型组和对照组比较,模拟转染组miR-130a mRNAp-AKT、p-GSK3β及p-c-Raf表达升高,TUNEL阳性细胞数量和PTEN mRNA表达降低(P<0.05);与对照组比较,模型组IL-10含量降低、IL-6和TNF-α含量升高(P<0.05);与模型组比较,模拟转染组IL-10含量升高、IL-6和TNF-α含量降低(P<0.05);与模拟对照组比较,仅miR-130a模拟组抑制了野生型-PTEN的相对荧光素酶活性(P<0.05)。结论m iR-130a可能通过影响PI3K/AKT轴和PTEN减轻缺血性卒中引起的神经功能缺损。

Objective To investigate the mechanism of miR-130a promoting the recovery of neurological function in ischemic stroke by regulating phosphatase and tensin homolog deleted on chromosome ten(PTEN).Methods Sixty male SD rats were randomly divided into control group(sham operation,n=20),model group(operation to establish ischemic stroke model,n=20)and simulated transfection group(after operation to establish ischemic stroke model,miR-130a simulated transfection was injected into the lateral ventricle of rats,n=20).The first and second day after operation were evaluated by nerve injury severity scale(MNSs)The degree of nerve defect at 10 and 25 days;The expression of mir-130a and PTEN was analyzed by RT-PCR;miR-130a targeted PTEN was detected by luciferase reporter gene.The levels of interleukin-6(IL-6),interleukin-10(IL-10)and tumor necrosis factor were compared by enzyme-linked immunosorbent assay-α(TNF-α)content of;TUNEL staining was used to detect the level of neuronal apoptosis in brain tissue,and Western blot was used to analyze phosphorylated protein kinase B(p-Akt)and phosphorylated glycogen synthase kinase-3β(-GSK3β)And phosphorylated proto oncogene serine threonine protein kinase(p-c-Raf).Results mir-130a mimicles can reduce the mNSS score of rats in the model group,and the expression of miR-130a can reduce the degree of functional damage of rat nerve cells.Compared with the control group,the expression of miR-130a mRNA in the brain tissue at the operation side of the model group was decreased,while the expression of PTEN mRNA was increased(P<0.05).Compared with the model group and the control group,the expression of miR-130a RNA in the brain tissue of the operation side of rats in the simulated transfection group was increased.Compared with the model group,the expression of PTEN mRNA in the simulated transfection group was decreased(P<0.05).Compared with the simulated control group,the miR-130a simulated group inhibited the relative luciferase activity of wild-type PTEN(P<0.05),and the luciferase reporter gene assay confirmed that miR-130a targeted PTEN.Compared with the control group,the contents of IL-10 in the cerebral tissue of the operative side of the model group were decreased,and the contents of IL-6 and TNF-αwere increased(P<0.05).Compared with the model group,the contents of IIL-10 in the simulated transfection group were increased,while the contents of IL-6 and TNF-αwere decreased.Compared with the control group,the number of TUNEL positive cells in the brain tissue section of the operation side of the model group was increased.Compared with the model group,the number of TUNEL positive cells in the simulated transfection group was decreased.Compared with the control group,the number of TUNEL positive cells in the brain tissue section of the operation side of the model group was increased.Compared with the model group,the number of TUNEL positive cells in the simulated transfection group was decreased.Compared with the control group,the protein expressions of p-Akt,p-GSK3βand p-c-Raf(Ser338)in the homogenate of cerebral tissue on the operation side of model group were decreased(P<0.05).Compared with the model group,the protein expressions of p-Akt,p-GSK3βand p-c-Raf in the simulated transfection group were increased(P<0.05),suggesting that mir-130a promoted the occurrence of PI3K/AKT pathway.Conclusion miR-130a can prevent neurological deficit caused by ischemic stroke by affecting PI3K/AKT axis and PTEN.