LncRNA DANCR靶向miR-3646减轻缺氧诱导的大鼠心肌细胞系H9c2的损伤

doi:10.16352/j.issn.1001-6325.2023.01.0144

摘要/Abstract

摘要： 目的研究长链非编码RNA(lncRNA)分化拮抗非蛋白编码RNA(DANCR)在心肌缺血中的作用与微小RNA-3646(miR-3646)的靶向调控关系。方法大鼠心肌细胞系H9c2分为6组:对照组、缺氧组、pc-DANCR(DANCR过表达)组、pc-NC(DANCR过表达对照)组、miR-3646 mimic(miR-3646模拟物)组、mimic NC(miR-3646模拟物对照)组及pc-DANCR+miR-3646 mimic组。RT-qPCR检测lncRNA DANCR及miR-3646表达以判定转染效果;流式细胞测量术检测细胞凋亡率;CCK-8法检测细胞生存率;电镜观察H9c2细胞结构损伤;Western blot检测活化半胱氨酸天门冬氨酸蛋白酶-3(cleaved caspase-3)和血红素加氧酶-1(Ho-1)表达。双荧光素酶实验验证miR-3646与lncRNA DANCR和Ho-1的靶向关系。RNA干扰Ho-1(ShHo-1)与pc-DANCR共转染H9c2细胞,检测细胞生存率。结果与对照组相比,缺氧组H9c2细胞损伤、凋亡严重,生存率及lncRNA DANCR表达降低,miR-3646表达升高(P<0.05)。与缺氧组相比,pc-DANCR组H9c2细胞损伤、凋亡减轻,细胞生存率升高,Ho-1表达升高,miR-3646表达降低(P<0.05)。miR-3646模拟物组H9c2细胞损伤和凋亡进一步加重,且miR-3646模拟物可减弱pc-DANCR的抗损伤及抗凋亡作用(P<0.05)。干扰Ho-1可减弱lncRNA DANCR对缺氧诱导的H9c2细胞生存的促进作用(P<0.05)。结论 LncRNA DANCR可靶向下调miR-3646表达减轻缺氧诱导的H9c2细胞损伤。

关键词: 长链非编码RNA, 分化拮抗非蛋白编码RNA, 微小RNA-3646, 心肌细胞, 缺氧诱导

Abstract: Objective To study the role of long non-coding RNA (lncRNA) differentiation antagonizing non-coding RNA (DANCR) in myocardial ischemia and the targeted regulation of microRNA-3646 (miR-3646). Methods H9c2 cells(rat cardiomyocytes cell line)were separated into 6 groups: control group, anoxia group, pc-DANCR (DANCR over-expression) group, pc-NC (DANCR over-expression control) group, miR-3646 mimic (miR-3646 mimicry) group, mimic NC (miR-3646 mimic control) group, and pc-DANCR+miR-3646 mimic group; RT-qPCR method was implemented to measure the expression of lncRNA DANCR and miR-3646 to determine the transfection effect; Flow cytometry was performed to measure the rate of apoptosis; CCK-8 assay was implemented to cell measure survival; Electron microscopy was applied to observe the structural injury of H9c2 cells; Western blot was performed to measure the expression of activated cleaved caspase-3 (cleaved caspase-3) and heme oxygenase-1 (Ho-1). Dual-luciferase experiments were performed to verify the targeting relationship of miR-3646 with lncRNA DANCR and Ho-1. RNA interference Ho-1 (ShHo-1) and pc-DANCR were co-transfected into H9c2 cells, and the cell viability was measured. Results Compared with the control group, the injury and apoptosis of H9c2 cells in the anoxia group were serious, the survival rate and the expression of lncRNA DANCR were decreased, and the expression of miR-3646 was increased (P<0.05). Compared with the anoxia group, the injury and apoptosis of H9c2 cells in the pc-DANCR group were alleviated, the survival rate was increased, the expression of anti-injury protective factor Ho-1 was increased, and the expression of miR-3646 was decreased(P<0.05). The injury and apoptosis of H9c2 cells in the miR-3646 mimic group were further aggravated, and miR-3646 mimic was able to attenuate the anti-injury and anti-apoptotic effects of pc-DANCR (P<0.05). There was a targeted regulatory relationship between lncRNA DANCR, Ho-1 and miR-3646. Interfering with Ho-1 attenuated the promoting effect of lncRNA-DANCR on the survival of H9c2 cells induced by anoxia (P<0.05). Conclusions LncRNA DANCR can down-regulate the expression of miR-3646 and promote the expression of Ho-1 to alleviate anoxia-induced H9c2 cells injury and improve cell survival.

Key words: long non-coding RNA, differentiation antagonist non-coding RNA, microRNA-3646, cardiomyocytes, anoxia induction

中图分类号:

R541

邱立彬, 张新才, 赵倩. LncRNA DANCR靶向miR-3646减轻缺氧诱导的大鼠心肌细胞系H9c2的损伤[J]. 基础医学与临床, 2023, 43(1): 144-151.

QIU Libin, ZHANG Xincai, ZHAO Qian. LncRNA DANCR reduces anoxia-induced rat myocardial cell line H9c2 injury by targeting miR-3646[J]. Basic & Clinical Medicine, 2023, 43(1): 144-151.

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