微小RNA-423-5p靶向乙醛脱氢酶2减轻脂多糖诱导的人脐静脉血管内皮细胞损伤

doi:10.16098/j.issn.0529-1356.2020.06.010

摘要/Abstract

摘要：

目的探讨微小RNA-423-5p(miR-423-5p)对脂多糖（LPS）诱导血管内皮细胞损伤的保护及作用机制。方法用1 mg/L LPS诱导人脐静脉血管内皮细胞（HUVECs）24 h，Real-time PCR和Western blotting检测细胞中miR-423-5p和乙醛脱氢酶2（ALDH2）的表达。通过转染anti-miR-423-5p和pcDNA-ALDH2下调miR-423-5p和上调ALDH2表达，流式细胞术检测细胞凋亡率，Western blotting检测凋亡相关蛋白Bcl-2和Bax的表达，并用ELISA试剂盒检测LPS诱导后细胞上清液中白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）的含量；双荧光素酶报告系统验证miR-423-5p与ALDH2的调控关系。结果与对照相比，LPS可诱导HUVECs凋亡和损伤，使HUVECs中miR-423-5p、Bax表达量及IL-6和TNF-α分泌量均显著升高（P<0.05），ALDH2的mRNA和蛋白表达量及Bcl-2量显著降低（P<0.05）；下调mi-423-5p表达和过表达ALDH2均可减轻LPS诱导的HUVECs损伤并抑制细胞凋亡；miR-423-5p靶向负调控ALDH2的表达；抑制ALDH2表达逆转了下调miR-423-5p表达对LPS诱导的HUVECs损伤的作用。结论下调miR-423-5p表达可靶向ALDH2减轻LPS对HUVECs的损伤并抑制细胞凋亡。

关键词: 脐静脉血管内皮细胞, 脂多糖, 微小RNA-423-5p, 乙醛脱氢酶2, 实时定量聚合酶反应, 流式细胞术, 人

Abstract:

Objective To investigate the protective effect of microRNA-423-5p(miR-423-5p) on injury of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide(LPS) and its potential mechanism. Methods The HUVECs were induced by 1 mg/L LPS for 24 hours, then expression levels of miR-423-5p and aldehyde dehydrogenasel 2 (ALDH2) in HUVECs induced by LPS were determined by Real-time PCR and Western blotting. The miR-423-5p was down-regulated and ALDH2 was up-regulated by transfected with anti-miR-423-5p and pcDNA-ALDH2. The apoptotic rate of HUVECs was detected by flow cytometry, and the expression levels of Bcl-2 and Bax were measured by Western blotting. The levels of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in supernatant of HUVECs induced by LPS were determined by the kits. The relationship between miR-423-5p and ALDH2 was validated by dual-luciferase reporter assay system. Results Compared with the control group, LPS induced apoptosis and injury of HUVECs. After LPS induction, the levels of miR-423-5p, Bax, IL-6 and TNF-α in HUVECs increased significantly (P<0.05), and the levels of Bcl-2, ALDH2 mRNA and ALDH2 protein decreased remarkably (P<0.05). Either down-regulation of miR-423-5p or over-expression of ALDH2 alleviated the injury of HUVECs induced by LPS and inhibited apoptosis. MiR-423-5p targeted and negatively regulated the expression of ALDH2. Inhibition of ALDH2 reversed the effects of miR-423-5p down-regulation on the injury of HUVECs induced by LPS.

Conclusion MiR-423-5p alleviates the injury of HUVECs induced by LPS and inhibits apoptosis by targeting ALDH2.

Key words: Umbilical vein endothelial cell, Lipopolysaccharide, MicroRNA-423-5p, Aldehyde dehydrogenase 2, Real-time PCR, Flow cytometry, Human

中图分类号:

R543.6

刘文芳. 微小RNA-423-5p靶向乙醛脱氢酶2减轻脂多糖诱导的人脐静脉血管内皮细胞损伤[J]. 解剖学报, 2020, 51(6): 868-876.

LIU Wen-fang. MicroRNA-423-5p reducing the injury of human umbilical vascular endothelial cells induced by lipopolysaccharide through targeting aldehyde dehydrogenase 2[J]. Acta Anatomica Sinica, 2020, 51(6): 868-876.

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