摘要
目的:探讨miRNA-210调控缺氧诱导因子-1α（HIF-1α）在血管性认知功能障碍（VCI）中的作用及分子机制。方法:60只雄性220~250 g大鼠随机分为4组，假手术组（Sham组）、VCI模型组（VCI组）、VCI模型+侧脑室注射miRNA-210 mimic组（VCI+miRNA-210 mimic组）和VCI模型+侧脑室注射miRNA-210 mimic对照序列组（VCI+miRNA-210 NC组）。应用四血管法建立VCI大鼠模型；侧脑室微量注射miRNA-210 mimic上调miRNA-210表达；7 d、14 d和30 d应用Morris水迷宫评价各组大鼠的学习记忆能力；应用实时荧光定量PCR法检测海马组织miRNA-210的表达；应用免疫印迹法检测海马组织HIF-1α和核转录因子-κB（NF-κB）的表达；应用ELISA法检测海马组织中肿瘤坏死因子-α（TNF-α）的表达，构建HIF-1α的3'非翻译区（3'UTR）双荧光素酶报告基因载体，验证miRNA-210调控HIF-1α的分子机制。采用t检验或Dunnett's T3法比较组间差异。结果:VCI模型大鼠出现认知功能障碍，7 d、14 d和30 d VCI组逃避潜伏期较Sham组、VCI+miRNA-210 mimic组显著升高，穿越原平台位置次数显著降低，差异均具有统计学意义（P均＜0.05）。与Sham组比较，VCI组及VCI+miRNA-210 NC组海马组织中HIF-1α和NF-κB的表达量增高，炎症因子TNF-α的释放增加，差异均具有统计学意义（P均＜0.05）；与VCI组比较，VCI+miRNA-210 mimic组海马组织中HIF-1α和NF-κB的表达量降低，炎症因子TNF-α的释放减少，差异均具有统计学意义（P均＜0.05）。VCI模型大鼠在侧脑室注射miRNA-210 mimic后，分离海马原代细胞，共转染miRNA-210与野生型HIF-1α 3'UTR的组中荧光素酶活性较VCI模型大鼠显著降低，提示miRNA-210与HIF-1α存在靶向关系。结论:miRNA-210可通过靶向抑制HIF-1α的表达，减轻大鼠海马区域炎症，改善大鼠VCI。
Abstract
Objective:To investigate the role and molecular mechanism of miRNA-210 regulating hypoxia inducible factor-1α (HIF-1α) expression in vascular cognitive impairment (VCI).Methods:60 male 220-250 g rats were randomly divided into Sham operation group (Sham), Vascular cognitive impairment (VCI), Vascular cognitive impairment + miRNA-210 mimics group (VCI+miRNA-210 mimic) and Vascular cognitive impairment + mimics control group (VCI+ mimic control). The rat model of vascular cognitive impairment was established by four - vessel method. In order to up-regulated the expression of miRNA-210, the miRNA-210 mimic was injected into lateral ventricles. Morris water maze was used to evaluate the learning and memory ability of rats in each group on day 7,14 and 30 after VCI. The expression of miRNA-210 in the hippocampus was detected by qPCR.Western blotting was used to detect the expression levels of HIF-1α and NF-kB. The concentration of TNF-α in the hippocampus was detected by ELISA. The 3' untranslated region (3'UTR) dual luciferase reporter gene vector of HIF-1α was constructed to verify the molecular mechanism of miRNA-210 regulating HIF-1α. T test or Dunnett's T3 method were used to compare the differences among groups.Results:In VCI group, the cognitive impairment occurred, which the mean escape latency at 7, 14 and 30 days was significantly increased and the times of crossing the original platform was significantly decreased (P＜0.05). Compared with Sham group, the expressions of HIF-1α and NF-κB in the hippocampus of VCI group and VCI+miRNA-210 NC group were increased, and the release of inflammatory factor TNF-α was increased, with statistical significance（P＜0.05）. After injected miRNA-210 mimics into lateral ventricles, primary hippocampal cells were isolated.The activity of luciferase was significantly decreased in the co-transfected with miRNA-210 and wild-type HIF-1α 3’UTR group suggesting a target relationship between miRNA-210 and HIF-1α.Conclusion:MiRNA-210 can improve vascular cognitive impairment in rats by reducing inflammation in the hippocampus, and the mechanism may be related to the down-regulation of HIF-1α expression.
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