miR-140对缺血性脑卒中水通道蛋白4表达水平的影响

The effect of MiR-140 on expression level of Aquaporin 4 in ischemic stroke

目的探讨miR-140对缺血性脑卒中水通道蛋白4(Aquaporin4, AQP4)表达水平的影响。方法通过栓塞大鼠大脑中动脉24 h来建立大鼠大脑中动脉栓塞(MCAO)模型;构建包含有人和大鼠的miR-140结合位点的AQP4 mRNA 3′非翻译端(3′UTR)的双荧光素酶报告基因质粒,检测miR-140对AQP4 mRNA 3′UTR的靶向作用;培养乳鼠原代星形胶质细胞,建立体外原代星形胶质细胞氧糖剥离(OGD)模型,转染miR-140拟似物(mimics)和miR-140抑制剂(inhibitor)观察过表达和沉默miR-140对AQP4的调控作用;免疫印迹法(Western blot)检测AQP4的表达水平;荧光实时定量PCR(Real-time PCR)检测miR-140的表达水平。结果与假手术组比较,MCAO模型大鼠脑组织中miR-140表达水平降低(59.3±10.0)%(P<0.05),而AQP4蛋白表达水平升高(1.8±0.3)倍(P<0.05);与对照组比较,原代星形胶质细胞氧糖剥离(OGD)模型miR-140表达水平降低(51.6±15.3)%(P<0.05),而AQP4蛋白表达水平升高(1.7±0.4)倍(P<0.05);双荧光素酶报告基因检测显示miR-140拟似物能显著降低荧光素酶活性(P<0.05),miR-140抑制剂能显著升高荧光素酶活性(P<0.05);星形胶质细胞中过表达miR-140能够抑制AQP4蛋白表达,与对照组比较过表达miR-140组AQP4蛋白表达水平降低(37.7±16.9)%(P<0.05),沉默miR-140可诱导AQP4蛋白表达水平升高(1.5±0.1)倍(P<0.05)。结论缺血性脑卒中脑组织及星形胶质细胞中miR-140表达水平降低,AQP4表达水平升高;miR-140能够靶向作用于AQP4 mRNA 3′非翻译区来抑制AQP4蛋白的表达。

Objective To investigate the effect of miR-140 on Aquaporin4(AQP4) expression level in ischemic stroke. Methods The rat middle cerebral artery was embolized for 2 hours to establish the cerebral infarction model of middle cerebral artery occlusion(MCAO). The dual luciferase reporter plasmid containing the binding site of miR-140 was constructed and used to verify whether miR-140 could target to the 3′ untranslated region(3′UTR) of AQP4. Primary astrocyte was cultured and primary astrocyte oxygen-glucosedeprivation(OGD) model was established in vitro, miR-140 mimics and inhibitors were transfectedfor over expression or down-regulation of miR-140, and the effect of miR-140 on regulating expression of AQP4 was detected. The expression level of AQP4 was detected by Western blotting. The expression level of miR-140 was detected by real-time PCR. Results Compared with the sham group, the expression of AQP4 protein in MCAO rats was increased by(59.3±10.0)%(P<0.05), and the expression of miR-140 was decreased by(1.8±0.30) folds(P< 0.05). Compared with the negative NC group, the expression of AQP4 protein was increased(1.7±0.4) folds(P<0.05) and the expression of miR-140 was decreased(51.6±15.3)%(P<0.05) in OGD model. The dual luciferase reporter gene assay showed that miR-140 mimics significantly decreased the activity of luciferase(P<0.05) and miR-140 inhibitor significantly increased the activity of luciferase(P<0.05). Over expression of miR-140 in astrocytes significantly decreased AQP4 protein expression by(37.7±16.9)%(P<0.05). Silencing miR-140 increased AQP4 protein expression by(1.5±0.1) folds(P<0.05). Conclusion Down-regulation of miR-140 wass associated with increased expression of AQP4 in cerebrum and astrocytes of ischemic stroke. miR-140 regulated the expression of AQP4 by targeting the 3′UTR of mRNA.