长链非编码RNA alpha-2-巨球蛋白反义RNA 1靶向微小RNA-106b-5p调控氧化型低密度脂蛋白诱导的人脑微血管内皮细胞损伤

doi:10.16098/j.issn.0529-1356.2023.03.010

解剖学报 ›› 2023, Vol. 54 ›› Issue (3): 319-327.doi: 10.16098/j.issn.0529-1356.2023.03.010

长链非编码RNA alpha-2-巨球蛋白反义RNA 1靶向微小RNA-106b-5p调控氧化型低密度脂蛋白诱导的人脑微血管内皮细胞损伤

李薇^1* 王丽² 汪志华³刘庆春¹韩荣胜4

1. 青海省第五人民医院神经内科，西宁810000; 2. 青海省第五人民医院内分泌科，西宁 810000; 3. 青海心脑血管病专科医院影像科，西宁 810012; 4. 青海省第五人民医院心内科，西宁 810000

收稿日期:2021-01-28 修回日期:2022-05-09 出版日期:2023-06-06 发布日期:2023-06-06
通讯作者: 李薇 E-mail:vks081@163.com

Long non-coding RNA alpha-2-macroglobulin antisense RNA 1 regulating oxidized low density lipoprotein-induced human brain microvascular endothelial cell damage by targeting microRNA-106b-5p

LI Wei^1* WANG Li² WANG Zhi-hua³ LIU Qing-chun¹ HAN Rong-sheng⁴

1.Department of Neurology, the Fifth People’s Hospital of Qinghai Province, Xining810000, China; 2.Department of Endocrinology, the Fifth People’s Hospital of Qinghai Province, Xining810000, China; 3.Department of Imaging, Qinghai Cardiovascular and Cerebrovascular Disease Hospital, Xining810012, China; 4.Department of Cardiology, the Fifth People’s Hospital of Qinghai Province, Xining810000, China

Received:2021-01-28 Revised:2022-05-09 Online:2023-06-06 Published:2023-06-06
Contact: LI Wei E-mail:vks081@163.com

摘要/Abstract

摘要：

目的探讨长链非编码RNA (lncRNA）alpha-2-巨球蛋白反义RNA 1（A2M-AS1）靶向微小RNA（miR）-106b-5p对氧化型低密度脂蛋白（ox-LDL）诱导的人脑微血管内皮细胞损伤的影响。方法用ox-LDL诱导人脑微血管内皮细胞设为ox-LDL组，正常培养细胞为对照(Ctrl)组；A2M-AS1过表达（pcDNA-A2M-AS1组）、空载体（pcDNA组）、miR-106b-5p抑制剂（anti-miR-106b-5p组）、阴性对照（anti-miR-NC组）、pcDNA-A2M-AS1与对照mimic NC（miR-NC组）、pcDNA-A2M-AS1与miR-106b-5p模拟物（miR-106b-5p mimics组）转染细胞后加ox-LDL处理，n=9；Real-time PCR检测A2M-AS1与miR-106b-5p表达；试剂盒检测丙二醛（MDA）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）水平；流式细胞术及TUNEL法检测细胞凋亡；双荧光素酶报告基因实验检测A2M-AS1与miR-106b-5p靶向关系；Western blotting检测Bcl-2和Bax蛋白表达量。结果与Ctrl组比较，ox-LDL组A2M-AS1表达水平、SOD和CAT活性、Bcl-2蛋白水平降低，miR-106b-5p表达水平、MDA水平、凋亡率、Bax蛋白水平升高（P＜0.05）；过表达A2M-AS1或干扰miR-106b-5p降低ox-LDL诱导细胞后MDA水平、凋亡率与Bax蛋白水平，升高SOD、CAT活性和Bcl-2蛋白水平（P＜0.05）；A2M-AS1靶向miR-106b-5p；上调miR-106b-5p逆转过表达lncRNA A2M-AS1对ox-LDL诱导的人脑微血管内皮细胞损伤的作用。结论 A2M-AS1通过靶向miR-106b-5p减轻ox-LDL诱导的人脑微血管内皮细胞损伤。

关键词:

长链非编码RNA, Alpha-2-巨球蛋白反义RNA 1, 微小RNA-106b-5p, 氧化型低密度脂蛋白, 氧化应激, 实时定量聚合酶链反应, 流式细胞术, 人脑微血管内皮细胞

Abstract:

Objective To investigate the effect of long non-coding RNA(lncRNA) alpha-2-macroglobulin antisense RNA 1 (A2M-AS1) targeting microRNA (miR）-106b-5p on oxidized low-density lipoprotein (ox-LDL)-induced injury of human brain microvascular endothelial cells. Methods Human brain microvascular endothelial cells (ox-LDL group) were induced by ox-LDL, normal cultured cells were control group (Ctrl); A2M-AS1 overexpression (pcDNA-A2M-AS1 group), empty vector (pcDNA group), miR-106b-5p inhibitor (anti-miR-106b-5p group), negative control (anti-miR-NC group), pcDNA-A2M-AS1 with control mimic NC (miR-NC group), pcDNA-A2M-AS1 with miR-106b-5p mimic (miR-106b-5p mimics group) were transfected into cells and treated with ox-LDL, n=9. Real-time PCR was used to detect the expression levels of A2M-AS1 and miR-106b-5p; Kits were used to detect malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT)); Flow cytometry and TUNEL detected apoptosis; Dual luciferase reporter gene assay detected A2M-AS1 and miR-106b-5p targeting; Western blotting detected Bcl-2 and Bax protein expression. Results Compared with the Ctrl group, the expression level of A2M-AS1 in the ox-LDL group decreased, and the activity of SOD and CAT and the protein level of Bcl-2 decreased (P＜0.05), while the expression level of miR-106b-5p and the level of MDA increased (P<0.05), and the rate of apoptosis and the protein level of Bax increased (P<0.05). Overexpressing A2M-AS1 or interfering with miR-106b-5p decreased the MDA level, apoptosis rate and Bax protein level after ox-LDL-induced cells, and increased SOD, CAT activity and Bcl-2 protein level (P＜0.05). A2M-AS1 targeted miR-106b-5p; up-regulation of miR-106b-5p reversed the effect of overexpressed lncRNA A2M-AS1 on ox-LDL-induced injury of human brain microvascular endothelial cells (P<0.05). Conclusion A2M-AS1 attenuates ox-LDL-induced injury of human brain microvascular endothelial cells by targeting miR-106b-5p.

Key words:

Long non-coding RNA, Alpha-2-macroglobulin antisense RNA 1, MicroRNA-106b-5p, Oxidized low density lipoprotein, Oxidative stress, Real-time PCR, Flow cytometry, Human brain microvascular endothelial cell

中图分类号:

R657.51

李薇王丽汪志华刘庆春韩荣胜 . 长链非编码RNA alpha-2-巨球蛋白反义RNA 1靶向微小RNA-106b-5p调控氧化型低密度脂蛋白诱导的人脑微血管内皮细胞损伤[J]. 解剖学报, 2023, 54(3): 319-327.

LI Wei WANG Li WANG Zhi-hua LIU Qing-chun HAN Rong-sheng .

Long non-coding RNA alpha-2-macroglobulin antisense RNA 1 regulating oxidized low density lipoprotein-induced human brain microvascular endothelial cell damage by targeting microRNA-106b-5p

[J]. Acta Anatomica Sinica, 2023, 54(3): 319-327.

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长链非编码RNA alpha-2-巨球蛋白反义RNA 1靶向微小RNA-106b-5p调控氧化型低密度脂蛋白诱导的人脑微血管内皮细胞损伤

Long non-coding RNA alpha-2-macroglobulin antisense RNA 1 regulating oxidized low density lipoprotein-induced human brain microvascular endothelial cell damage by targeting microRNA-106b-5p

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