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.

参考文献

[1] 李思源, 刘振兵. 氧化应激在心肌缺血再灌注损伤中的研究进展[J]. 山东医药, 2021, 61: 92-95.
[2] 曾玉玲, 李世阁, 占德进. 参麦注射液促进心肌梗死模型大鼠心肌血管新生[J]. 基础医学与临床, 2021, 41: 1624-1628.
[3] 曹扬, 封佳曦, 鲁明, 等. LncRNA DANCR作为常见恶性肿瘤潜在预后因子的Meta分析[J]. 医学综述, 2020, 26: 2020-2025+2030.
[4] Qiu L, Zhao Q, Dai L, et al. Long non-coding RNA DANCR alleviates hypoxia-caused H9c2 cells damage through up regulation of HIF-1α[J]. Artif Cell Nanomed B, 2020, 48: 533-541.
[5] Kang X, Cao S, Ji Z, et al. miR-3646 promotes vascular inflammation and augments vascular smooth muscle cell proliferation and migration in progression of coronary artery disease by directly targeting RHOH[J]. Int J Clin Exp Pathol, 2018, 11: 5830-5839.
[6] 王苗, 王裕岱, 黄玉冰, 等. NPHP3-AS1通过调控miR-30a-5p影响缺氧诱导的心肌细胞增殖及凋亡的研究[J]. 中国循证心血管医学杂志, 2020, 12: 918-923.
[7] 李慰, 翁立斌, 李学和, 等. LncRNA DANCR通过调控miR-214-5p在宫颈癌细胞增殖及侵袭中的作用研究[J].中国临床药理学杂志, 2021, 37: 1672-1675.
[8] Li J, Xie J, Wang YZ, et al. Overexpression of lncRNA Dancr inhibits apoptosis and enhances autophagy to protect cardiomyocytes from endoplasmic reticulum stress injury via sponging microRNA-6324[J]. Mol Med Rep, 2021, 23: 1-10.
[9] 李骞, 李秀研, 林丽丽, 等. LncRNA DANCR靶向miR-423-5p调控ox-LDL诱导的血管内皮细胞凋亡,炎症反应[J]. 中国免疫学杂志, 2021, 37: 2187-2193.
[10] 殷卫兵, 杨芳, 郭建军, 等.长链非编码RNA MEF2C-AS1通过下调miR-3646的表达抑制乳腺癌细胞的增殖、迁移和侵袭的功能机制研究[J].中华生物医学工程杂志,2019,25: 678-684.
[11] Waldman M, Arad M, Abraham NG, et al. The peroxisome proliferator-activated receptor-gamma coactivator-1α-heme oxygenase 1 axis, a powerful antioxidative pathway with potential to attenuate diabetic cardiomyopathy[J]. Antioxid Redox Signal, 2020, 32: 1273-1290.
[12] 赵荫涛, 杨海波, 刘源,等. 螺内酯对缺氧/复氧处理的人心肌细胞HO-1/Nrf2/SIRT3信号通路及自噬的影响[J]. 中国急救医学, 2021, 41: 413-418.

[1]	郑红, 陈晓霞, 韩李周, 陈苗, 皇甫娟. 降低lncRNA-RMRP表达抑制人肺癌细胞系A549的增殖和侵袭[J]. 基础医学与临床, 2024, 44(7): 974-978.
[2]	闫霖, 陆珏秀, 罗颖, 刘先霞. BM-MSCs来源外泌体介导铁死亡减轻大鼠心肌细胞系H9c2缺氧/复氧损伤[J]. 基础医学与临床, 2023, 43(5): 771-776.
[3]	苏越, 梁琳慧, 何祥火. 长链非编码RNA亚细胞定位和功能的研究进展[J]. 基础医学与临床, 2023, 43(10): 1580-1584.
[4]	刘磊, 李世宝, 张好良. 沉默lnc-SLC2A12-10:1抑制人胃癌细胞系AGS增殖、迁移和侵袭[J]. 基础医学与临床, 2022, 42(7): 1071-1076.
[5]	郝相楠, 栾军军, 马聪, 张旖骁, 周华. 过表达miR-150的肾小管上皮细胞系HK-2中lncRNA的表达谱[J]. 基础医学与临床, 2022, 42(7): 1013-1019.
[6]	谢利华, 杨奇, 李雅倩, 谢心悦, 雷诗娟, 周琳. 转录因子Kaiso下调人乳腺癌细胞系中lncRNA MEG3的表达[J]. 基础医学与临床, 2022, 42(5): 758-762.
[7]	陈思, 杨富国, 彭红军, 曹卫乐, 王振宇. 丙泊酚减轻缺氧/复氧诱导的心肌细胞损伤[J]. 基础医学与临床, 2022, 42(4): 583-589.
[8]	杨晔, 党荣广, 张静, 董超, 宋雪晶. 肺癌组织中lncRNA ZFAS1和hsa-miR-372-3p的表达与患者恶性特征及预后的关系[J]. 基础医学与临床, 2022, 42(12): 1861-1866.
[9]	滕培英, 李静, 师玉露, 杨帆, 欧霞, 陈伟. 柯萨奇病毒B组5型感染的人恶性胚胎横纹肌肉瘤细胞系lncRNA表达谱分析[J]. 基础医学与临床, 2022, 42(1): 68-74.
[10]	宫丹丹, 隋春兴, 石晨, 李月, 姜晓东. 沉默miR-26a减轻缺血/再灌注大鼠心肌损伤[J]. 基础医学与临床, 2021, 41(9): 1285-1290.
[11]	蒙颖, 张正义, 赵嫄, 卢昌宏, 商波, 孙鹏飞, 张德奎. X线放射诱导大鼠心肌细胞系H9c2损伤中组蛋白乙酰化水平降低[J]. 基础医学与临床, 2021, 41(8): 1139-1143.
[12]	雷家秀, 朱雅萍, 张雨. 依托咪酯上调miR-290-5p减轻LPS诱导的大鼠心肌细胞系H9C2损伤[J]. 基础医学与临床, 2021, 41(6): 848-853.
[13]	赵俞乔, 苏志雷, 崔云甫, 关沧海, 姜兴明. 恶性肿瘤中HOTAIRM1的调控作用及机制[J]. 基础医学与临床, 2021, 41(2): 262-266.
[14]	尤旭, 朱小芳, 胡运鹏, 龚杨, 赵磊. 黄芪总皂苷对心衰大鼠心肌细胞凋亡和线粒体膜电位的影响[J]. 基础医学与临床, 2020, 40(9): 1218-1223.
[15]	胡雪停, 吴晓凤, 徐祥. 长链非编码RNA lnc-DC的生物信息学分析与验证[J]. 基础医学与临床, 2020, 40(10): 1328-1334.