长链非编码RNA牛磺酸上调基因1敲低通过调控miR-532-5p/Sox8轴保护心肌细胞免受缺氧/复氧诱导的损伤。

Long Noncoding RNA Taurine-Upregulated Gene 1 Knockdown Protects Cardiomyocytes Against Hypoxia/Reoxygenation-induced Injury Through Regulating miR-532-5p/Sox8 Axis.

作者信息

Cai Xinyong, Wang Shu, Hong Lang, Yu Songping, Li Bin, Zeng Hong, Yang Xu, Zhang Ping, Shao Liang

机构信息

Department of Cardiology, Jiangxi Provincial People's Hospital Affiliated to Nanchang University, Nanchang, China.

Department of Gerontology, The First Affiliated Hospital of Nanchang University, Nanchang, China.

出版信息

J Cardiovasc Pharmacol. 2020 Nov;76(5):556-563. doi: 10.1097/FJC.0000000000000895.

DOI:10.1097/FJC.0000000000000895

PMID:32833900

Abstract

BACKGROUND

Long noncoding RNA taurine-upregulated gene 1 (TUG1) has been reported to involve in the processing of cardiac ischemia/reperfusion injury after myocardial infarction. Thus, this study further investigates the underlying mechanisms of TUG1 in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury in vitro.

METHODS

Cell viability, apoptosis, and migration and invasion were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and transwell assay, respectively. Western blot was used to examine the levels of matrix metallopeptidase 9, matrix metallopeptidase 2, and sex determining region Y-box transcription factor 8 (Sox8) protein. Levels of lactate dehydrogenase, malondialdehyde, superoxide dismutase, and glutathione peroxidase were detected using commercial kits. Levels of TUG1, microRNA-532-5p (miR-532-5p), and Sox8 were detected by quantitative real-time polymerase chain reaction. The interaction between miR-532-5p and Sox8 or TUG1 was confirmed by dual-luciferase reporter and RNA immunoprecipitation assay.

RESULTS

H/R induced rat cardiomyocyte H9c2 injury by inhibiting cell viability, migration and invasion, promoting cell apoptosis, and stimulating oxidative stress. H/R-induced H9c2 injury upregulated the level of TUG1, and TUG1 knockdown alleviated H/R-induced cardiomyocyte injury. TUG1 directly bound to miR-532-5p, and miR-532-5p inhibition reversed the action of TUG1 knockdown on H/R-induced cardiomyocyte injury. Sox8 was a target of miR-532-5p, and miR-532-5p blunted H/R-induced cardiomyocyte injury by targeting Sox8. In addition, TUG1 knockdown inhibited H/R-induced Sox8 elevation through miR-532-5p in H9c2 cells.

CONCLUSION

TUG1 silence ameliorated H/R-induced cardiomyocytes injury through regulating miR-532-5p/Sox8 axis, suggesting a potential therapeutic target for preventing myocardial ischemia/reperfusion injury.

摘要

背景

据报道，长链非编码RNA牛磺酸上调基因1（TUG1）参与心肌梗死后心脏缺血/再灌注损伤的过程。因此，本研究进一步探讨TUG1在体外缺氧/复氧（H/R）诱导的心肌细胞损伤中的潜在机制。

方法

分别采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法、流式细胞术和Transwell法检测细胞活力、凋亡、迁移和侵袭。采用蛋白质免疫印迹法检测基质金属蛋白酶9、基质金属蛋白酶2和性别决定区Y盒转录因子8（Sox8）蛋白水平。使用商业试剂盒检测乳酸脱氢酶、丙二醛、超氧化物歧化酶和谷胱甘肽过氧化物酶水平。通过定量实时聚合酶链反应检测TUG1、微小RNA-532-5p（miR-532-5p）和Sox8的水平。通过双荧光素酶报告基因和RNA免疫沉淀实验证实miR-532-5p与Sox8或TUG1之间的相互作用。

结果

H/R通过抑制细胞活力、迁移和侵袭、促进细胞凋亡以及刺激氧化应激诱导大鼠心肌细胞H9c2损伤。H/R诱导的H9c2损伤上调了TUG1水平，而TUG1基因敲低减轻了H/R诱导的心肌细胞损伤。TUG1直接与miR-532-5p结合，抑制miR-532-5p可逆转TUG1基因敲低对H/R诱导的心肌细胞损伤的作用。Sox8是miR-532-5p的靶标，miR-532-5p通过靶向Sox8减轻H/R诱导的心肌细胞损伤。此外，在H9c2细胞中，TUG1基因敲低通过miR-532-5p抑制H/R诱导的Sox8升高。