circDLPAG4/HECTD1 mediates ischaemia/reperfusion injury in endothelial cells via ER stress.
circDLPAG4/HECTD1 mediates ischaemia/reperfusion injury in endothelial cells via ER stress.
复制标题
circDLPAG4/HECTD1 通过 ER 应激介导内皮细胞缺血/再灌注损伤。
DOI：
10.1080/15476286.2019.1676114
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发表时间：
2020-02-01
期刊：
影响因子：
4.1
通讯作者：
Chao, Jie
中科院分区：
文献类型：
--
作者：
Chen, Lulu;Luo, Wei;Chao, Jie
背景：以细胞凋亡和迁移为特征的血管内皮细胞功能障碍在心血管疾病常见的缺血/再灌注损伤中起着至关重要的作用。最近的研究表明，环状RNA等非编码RNA在I/R损伤的细胞功能障碍中起作用，但具体机制尚不清楚。方法：以人脐静脉内皮细胞为模型。Western blotting(WB)和免疫细胞化学检测蛋白表达。使用CRISPR/Cas9系统、WB、细胞活力测定、Hoechst染色和三维迁移模型观察细胞功能的变化。采用实时荧光定量聚合酶链式反应和FISH技术，结合慢病毒转染法对CircRNAs和miRNAs的表达进行检测。结果：在HUVECs中，HUVECs中HECTD1的表达呈时间依赖性下降，细胞存活率下降约45%，细胞的凋亡和迁移增加，HECTD1的过表达减弱了这一作用。I/R诱导的内质网应激上调也可减弱HECTD1的过表达。此外，miR-143模拟物可抑制HECTD1的表达，而这种表达可通过CircDLGAP4的过表达而恢复，这为深入了解I/R诱导的HECTD1在内皮细胞功能障碍中的分子机制提供了线索。结论：CircDLGAP4和HECTD1在I/R诱导的内皮细胞功能障碍中起关键作用，为治疗心肌缺血的潜在治疗靶点提供了新的视角。
Background: Vascular endothelial cell dysfunction, characterized by cell apoptosis and migration, plays a crucial role in ischaemia/reperfusion (I/R) injury, a common aspect of cardiovascular diseases. Recent studies have suggested that non-coding RNAs, such as circular RNAs (circRNA), play a role in cell dysfunction in I/R injury, although the detailed mechanism is unclear.Methods: Human umbilical vein endothelial cells (HUVECs) were used for in vitro I/R model. Protein expression was detected by western blotting (WB) and immunocytochemistry. The CRISPR/Cas9 system, WB, cell viability assays, Hoechst staining and a 3D migration model were used to explore functional changes. RNA expression was evaluated using quantitative real-time PCR and a FISH assay combined with lentivirus transfection regulating circRNAs and miRNAs. A mouse myocardial I/R model using C57 mice was established to confirm the in vitro findings.Results: In HUVECs, I/R induced a significant time-dependent decrease in HECTD1 associated with an approximately 45% decrease in cell viability and increases in cell apoptosis and migration, which were attenuated by HECTD1 overexpression. I/R-induced upregulation of endoplasmic reticulum stress was also attenuated HECTD1 overexpression. Moreover, miR-143 mimics inhibited HECTD1 expression, which was restored by circDLGAP4 overexpression, providing insight as to the molecular mechanism of I/R-induced HECTD1 in endothelial cell dysfunction.Conclusion: Our results suggest a critical role for circDLGAP4 and HECTD1 in endothelial cell dysfunction induced by I/R, providing novel insight into potential therapeutic targets for the treatment of myocardial ischaemia.