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中华脑血管病杂志(电子版) ›› 2023, Vol. 17 ›› Issue (05) : 489 -494. doi: 10.11817/j.issn.1673-9248.2023.05.011

基础研究

MiRNA-210通过抑制HIF-1α的表达改善大鼠血管性认知功能障碍
刘感哲, 艾芬()   
  1. 430014 华中科技大学同济医学院附属武汉市中心医院神经内科
  • 收稿日期:2023-08-11 出版日期:2023-10-01
  • 通信作者: 艾芬
  • 基金资助:
    脑小血管病与脑磁共振及血清炎性因子相关性研究,武汉市卫生健康委员会科研项目(WX19C03)

MiRNA-210 improved vascular cognitive impairment in rats by inhibiting HIF-1α expression

Ganzhe Liu, Fen Ai()   

  1. Department of Neurology, the Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, China
  • Received:2023-08-11 Published:2023-10-01
  • Corresponding author: Fen Ai
引用本文:

刘感哲, 艾芬. MiRNA-210通过抑制HIF-1α的表达改善大鼠血管性认知功能障碍[J]. 中华脑血管病杂志(电子版), 2023, 17(05): 489-494.

Ganzhe Liu, Fen Ai. MiRNA-210 improved vascular cognitive impairment in rats by inhibiting HIF-1α expression[J]. Chinese Journal of Cerebrovascular Diseases(Electronic Edition), 2023, 17(05): 489-494.

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。

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.

图1 各组大鼠Morris水迷宫检测的结果比较。图a:术前1 d、术后第7、14、30天大鼠在Morris水迷宫实验中找到平台的时间(逃避潜伏期);图b:术后第7、14、30天大鼠在Morris水迷宫实验中穿越平台次数 注:Sham为假手术组;VCI为血管性认知功能障碍组;VCI+miRNA-210 mimic为血管性认知功能障碍组+miRNA-210 mimic处理组;VCI+miRNA-210 NC为血管性认知功能障碍+miRNA-210 mimic对照序列组。与Sham组比较:*P<0.05;与VCI组比较:#P<0.05
图2 各组大鼠海马区缺氧诱导因子1α(HIF-1α)及miRNA-210的表达水平。图a:免疫印迹法检测各组大鼠海马组织HIF-1α蛋白的表达量。图b:各组大鼠海马组织HIF-1α的相对表达量比较。图c:各组大鼠海马组织miRNA-210的相对表达量比较 注:Sham为假手术组;VCI为血管性认知功能障碍组;VCI+miRNA-210 mimic为血管性认知功能障碍组+miRNA-210 mimic处理组;VCI+miRNA-210 NC为血管性认知功能障碍+miRNA-210 mimic对照序列组。与Sham组比较:*P<0.05;与VCI组比较:#P<0.05
图3 各组大鼠海马组织核因子κB(NF-κB)及炎症因子肿瘤坏死因子α(TNF-α)的表达水平。图a:免疫印迹法检测各组大鼠海马组织NF-κB的含量;图b:各组大鼠海马组织NF-κB的相对表达量比较;图c:各组大鼠海马组织TNF-α的含量比较 注:Sham为假手术组;VCI为血管性认知功能障碍组;VCI+miRNA-210 mimic为血管性认知功能障碍组+miRNA-210 mimic处理组;VCI+miRNA-210 NC为血管性认知功能障碍+miRNA-210 mimic对照序列组。与Sham组比较:*P<0.05;与VCI组比较:#P<0.05
图4 原代海马细胞中各组荧光素酶活性相对值 注:HIF-1α为缺氧诱导因子1α,3'UTR为3'非翻译区;miRNA-210 NC为miRNA-210 mimic对照序列组,miRNA-210 mimic为miRNA-210 mimic处理组;WT HIF-1α 3'UTR为野生型HIF-1α 3'UTR转染组;MT HIF-1α 3'UTR为突变型HIF-1α 3'UTR转染组。与其他组比较:*P<0.05
图5 Targetscan预测缺氧诱导因子1α(HIF-1α)的3'UTR区与miRNA-210结合的序列
1
Pantoni L, Inzitari D. Hachinski's ischemic score and the diagnosis of vascular dementia: a review [J]. Ital J Neurol Sci, 1993, 14(7): 539-546.
2
Gorelick PB, Scuteri A, Black SE, et al. Vascular contributions to cognitive impairment and dementia: a statement for healthcare professionals from the american heart association/american stroke association [J]. Stroke, 2011, 42(9): 2672-2713.
3
罗璇, 岳卫东. 血管性认知功能障碍的研究进展 [J]. 现代生物医学进展, 2012, 12(21): 4156-4158.
4
Semenza GL. Hypoxia-inducible factors in physiology and medicine [J]. Cell, 2012, 148(3): 399-408.
5
Cao Y, Li Z, Ma L, et al. Isoflurane-induced postoperative cognitive dysfunction is mediated by hypoxia-inducible factor-1α-dependent neuroinflammation in aged rats [J]. Mol Med Rep, 2018, 17(6): 7730-7736.
6
Zeidman P, Maguire EA. Anterior hippocampus: the anatomy of perception, imagination and episodic memory [J]. Nat Rev Neurosci, 2016, 17(3): 173-182.
7
Tian T, Zhang Y, Wu T, et al. miRNA profiling in the hippocampus of attention-deficit/hyperactivity disorder rats [J]. J Cell Biochem, 2019, 120(3): 3621-3629.
8
Chan SY, Loscalzo J. MicroRNA-210: a unique and pleiotropic hypoxamir [J]. Cell Cycle, 2010, 9(6): 1072-1083.
9
侯外方, 张茂森, 张琳, 等. 血管性认知障碍动物模型的研究进展 [J]. 中国实验动物学报, 2021, 29(4): 542-552.
10
任家谋, 高玉梅, 吴昌学, 等. 血管性认知功能障碍大鼠胆碱酯酶及胆碱乙酰化酶的表达 [J]. 中国老年学杂志, 2020, 40(11): 2396-2401.
11
Fasanaro P, D'Alessandra Y, Di Stefano V, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3 [J]. J Biol Chem, 2008, 283(23): 15878-15883.
12
Chan YC, Banerjee J, Choi SY, et al. miR-210: the master hypoxamir [J]. Microcirculation, 2012, 19(3): 215-223.
13
Semenza GL, Agani F, Feldser D, et al. Hypoxia, HIF-1, and the pathophysiology of common human diseases [J]. Adv Exp Med Biol, 2000, 475: 123-130.
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