miR-582-5p调控Notch1减轻心肌缺血再灌注损伤的研究

王峰; 杨伟; 钱佃伦; 梅松; 王文杰; 冯科翔; 白向锋

doi:10.12259/j.issn.2095-610X.S20221207

miR-582-5p调控Notch1减轻心肌缺血再灌注损伤的研究

doi: 10.12259/j.issn.2095-610X.S20221207

1.
昆明医科大学实验动物学部，云南昆明　650500
2.
昆明医科大学第一附属医院麻醉科
3.
心脏外科，云南昆明　650032

基金项目: 云南省科技厅-昆明医科大学应用基础研究联合专项基金资助项目[2019FE001（-045）；202201AY070001-056]；云南省教育厅科学研究基金资助项目（2021J0230）

详细信息

作者简介:
王峰（1996～），男，江苏泰州人，在读博士研究生，主要从事神经系统疾病研究以及人类疾病动物模型的制作工作

通讯作者:
白向锋，E-mail：49976790@qq.com

中图分类号: R654.2
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出版历程
- 收稿日期: 2022-06-28
- 网络出版日期: 2022-12-05
- 刊出日期: 2022-12-25

miR-582-5p Regulating Notch1 to Reduce Myocardial Ischemia-reperfusion Injury

1.
Dept. of Laboratory Animal Science，Kunming Medical University，Kunming Yunnan 650500
2.
Dept. of Anesthesiology
3.
Dept. of Cardiac Surgery，The 1st Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650032，China

摘要

摘要: 目的检测小鼠心肌缺血再灌注和HL-1细胞HR模型后miR-582-5p和Notch1在其中的表达变化，以此探讨miR-582-5p调控Notch1保护心肌的作用。方法通过建立小鼠心肌缺血再灌注模型和HL-1细胞氧糖剥夺再复氧模型，使用qPCR检测miR-582-5p和Notch1在小鼠IR和细胞HR后的表达量，继而使用TargetScan网站预测miR-582-5p和Notch1的靶向结合位点，在细胞中转染miR-582-5p和Notch1的Inhibitor和Mimics，再次检测miR-582-5p和Notch1的表达变化，最后使用CCK-8实验检测HL-1细胞转染Inhibitor和Mimics后的细胞活力。结果在小鼠IR和HL-1细胞HR后，miR-582-5p表达明显升高（P < 0.01），Notch1表达显著下降（P < 0.001），而在转染Inhibitor后，miR-582-5p明显下调（P < 0.001），同时Notch1显著上调（P = 0.017），并且心肌细胞活力得到显著提高（P = 0.006），而在转染Mimics后结果完全相反。结论 miR-582-5p可以通过调控Notch1从而保护心肌以此来对抗心肌缺血缺氧。
- 心肌缺血再灌注 /
- 氧糖剥夺 /
- miR-582-5p /
- Notch1
Abstract: Objective To investigate the role of miR-582-5p in regulating Notch1 to protect myocardium, by detecting the expression changes of miR-582-5p and Notch1 after myocardial ischemia reperfusion and HR model of HL-1 cells in mice. Methods The mouse myocardial ischemia-reperfusion model and HL-1 cell OGD reoxygenation model were established. The expression of miR-582-5p and Notch1 in mouse IR and cell HR was detected by qPCR, and then the targeted binding sites of miR-582-5p and Notch1 were predicted by TargetScan. Inhibitor and mimics of miR-582-5p and Notch1 were transfected into cells, and the expression of miR-582-5p and Notch1 was detected again. Finally, CCK-8 assay was used to detect the cell viability of HL-1 cells transfected with inhibitor and mimics. Results After HR, the expression of miR-582-5p was significantly increased （P < 0.01） and the expression of Notch1 was significantly decreased in IR mouse and HL-1 cells （P < 0.001）. After transfection of inhibitor, the expression of miR-582-5p was significantly down-regulated （P < 0.001）, while Notch1 was significantly up-regulated （P = 0.017）, and the viability of cardiomyocytes was significantly increased （P = 0.006）, but the result was opposite after transfection of mimics. Conclusion miR-582-5p can protect myocardium against myocardial ischemia and hypoxia by regulating Notch1.
- Myocardial ischemia-reperfusion /
- Oxygen and glucose deprivation /
- miR-582-5p /
- Notch1

HTML全文

图 1 miR-582-5p和Notch1在心肌缺血再灌注小鼠心脏组织和HL-1细胞OGD模型中的表达

A：miR-582-5p在MRI小鼠心脏组织中的相对表达量（n = 5）； B：miR-582-5p在OGD模型HL-1细胞中的相对表达量（n = 3）；C：Notch1在MRI小鼠心脏组织中的相对表达量（n = 5）；D：Notch1在OGD模型HL-1细胞中的相对表达量（n = 3）。^**P < 0.01，^***P < 0.001。

Figure 1. Expression of miR-582-5p and Notch 1 in cardiac tissue and HL-1 cell OGD model of myocardial ischemia-reperfusion mice

下载: 全尺寸图片幻灯片

图 2 miR-582-5p可靶向Notch1并调控其表达

A：miR-582-5p与Notch1结合位点预测；B：miR-582-5p转染inhibitor和mimics后的相对表达量（n = 3）；C：Notch1转染inhibitor和mimics后的相对表达量（n = 3）；D：WB检测转染inhibitor后Notch1的表达及定量图（n = 3）。E：WB检测转染mimics后Notch1的表达及定量图（n = 3）。^*P < 0.05，^**P < 0.01，^***P < 0.001。

Figure 2. miR-582-5p targets Notch 1 and regulates its expression

下载: 全尺寸图片幻灯片

图 3 HL-1细胞活力的检测

A：正常HL-1细胞转染miR-582-5p的inhibitor和mimics后的细胞活力（n = 6）；B：HL-1细胞在OGD模型后复氧下转染miR-582-5p的inhibitor和mimics后的细胞活力（n = 6）。 ^**P < 0.01，^***P < 0.001。

Figure 3. Detection of HL-1 cell viability

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

基因名称	上游引物序列（5′- 3′）	下游引物序列（5′- 3′）
GAPDH	CTCGCCTTTGCCGATCC	GAATCCTTCTAGCCCATGCC
Notch1	GATGGCCtCAATGGGTACAAG	TCGTTGTTGTTGATGTCACAGT
miR-582-5p	通用引物	ATACAGTTGTTCAACCAGTTAC
U6	通用引物	ATGGACTATCATATGCTTACCGTA

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