lncRNA NEAT1调控miR-206对缺氧复氧大鼠心肌细胞氧化应激损伤和凋亡的影响

Effects of lncRNA NEAT1 on oxidative stress injury and apoptosis of hypoxic-reoxygenated rat cardiomyocytes by regulating miR-206

目的探讨长链非编码RNA(lncRNA)NEAT1调控miR-206对缺氧复氧诱导的心肌细胞氧化应激损伤和凋亡的影响和机制。方法体外培养H9c2心肌细胞,构建心肌细胞缺氧复氧模型。实时荧光定量PCR(RT-qPCR)检测缺氧复氧诱导后lncRNA NEAT1和miR-206的表达水平。将lncRNA NEAT1小干扰RNA(si-lncRNA NEAT1)、miR-206模拟物(miR-206 mimics)分别转染H9c2细胞,缺氧复氧诱导后,检测细胞中丙二醛(MDA)和活性氧(ROS)含量以及细胞上清液中乳酸脱氢酶(LDH)活性,MTT法检测细胞存活率,流式细胞术检测细胞凋亡,Western blot检测含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved Caspase-3)和cleaved Caspase-9蛋白表达。利用荧光素酶报告基因实验以及RT-qPCR验证lncRNA NEAT1和miR-206的靶向结合关系。结果缺氧复氧诱导后H9c2细胞中lncRNA NEAT1的表达显著升高,miR-206的表达显著降低(P<0.05)。转染si-lncRNA NEAT1或miR-206 mimics处理缺氧复氧H9c2细胞,细胞存活率显著升高,MDA、ROS含量以及LDH活性显著降低,cleaved Caspase-3和cleaved Caspase-9蛋白的表达显著降低,细胞凋亡率显著降低(P<0.05)。lncRNA NEAT1靶向miR-206并负调控miR-206表达。抑制miR-206部分逆转沉默lncRNA NEAT1对缺氧复氧诱导的心肌细胞氧化损伤和凋亡的影响(P<0.05)。结论沉默lncRNA NEAT1通过上调miR-206可减轻缺氧复氧诱导的心肌细胞氧化应激损伤和细胞凋亡,进而发挥心肌保护作用。

Aim To investigate the effect and mechanism of lncRNA NEAT1 on hypoxia/reoxygenation induced oxidative stress injury and apoptosis of cardiomyocytes by regulating miR-206. Methods H9 c2 cardiomyocytes were cultured in vitro to construct cardiomyocyte hypoxia/reoxygenation model. Real-time quantitative PCR(RT-qPCR) was used to detect the expression of lncRNA NEAT1 and miR-206 after hypoxia/reoxygenation induction. LncRNA NEAT1 small interference RNA(si-lncRNA NEAT1) and miR-206 mimics(miR-206 mimics) were transfected into H9 c2 cell, respectively. After hypoxia/reoxygenation induction, the kit was used to detect the content of malondialdehyde(MDA) and reactive oxygen species(ROS) in the cell and the content of lactate dehydrogenase(LDH) in the cell supernatant, MTT assay was used to detect cell activity, flow cytometry was used to detect cell apoptosis, and Western blot was used to detect the expression of cleaved Caspase-3 and cleaved Caspase-9 proteins. The luciferase reporter assay and RT-qPCR were used to verify the targeted binding relationship between lncRNA NEAT1 and miR-206. Results After the induction of hypoxia/reoxygenation, the expression of lncRNA NEAT1 in H9 c2 cell was significantly increased, while the expression of miR-206 was significantly decreased(P<0.05). After transfection with si-lncRNA NEAT1 or miR-206 mimics, the survival rate of H9 c2 cell was significantly increased, MDA, ROS contents and LDH activity were significantly reduced, and the expression of cleaved Caspase-3 and cleaved Caspase-9 proteins were significantly reduced, and the apoptosis rate was significantly reduced(P<0.05). lncRNA NEAT1 targets miR-206 and negatively regulates miR-206 expression. Inhibition of miR-206 partially reversed the effects of silencing lncRNA NEAT1 on oxidative damage and apoptosis of cardiomyocytes induced by hypoxia/reoxygenation(P<0.05). Conclusion Silencing lncRNA NEAT1 could reduce hypoxia/reoxygenation-induced oxidative stress injury and apoptosis of cardiomyocytes by up-regulating miR-206, and then exert myocardial protection.