Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China.
Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China.
Exp Cell Res. 2020 Oct 1;395(1):112172. doi: 10.1016/j.yexcr.2020.112172. Epub 2020 Jul 16.
During the process of myocardial ischemia-reperfusion injury (MIRI), the intracellular Ca concentration ([Ca]) continues to increase, leads to the cardiomyocyte apoptosis and eventually causes myocardial damage, while the upstream regulation mechanism of calcium overload is still unknown. This study focuses on the role of miR-219a-2 in MIRI and aims to elaborate its regulatory mechanism on calcium overload that occurs during MIRI.
The expression of miR-219a-2 was determined in the heart tissues of MIRI mice by qRT-PCR. The [Ca] was measured by fluo-3 using a fluorescence microplate reader. The expression of hypoxiainducible factor 1α (HIF1α) and NR1, the obligatory subunit of N-methyl-d-aspartate receptor 1 (NMDAR), were measured by qRT-PCR and western blot. The luciferase reporter assay was used to confirm the interplay between miR-219a-2 and HIF1α and the interplay between HIF1α and NR1. The cell apoptosis was measured by the expression level of B-cell lymphoma 2 interacting mediator of cell death (Bim) and the number of TUNEL-positive cells. The myocardial infarct size of mice was measured by TTC/Evans Blue staining.
MiR-219a-2 was down-regulated in the heart tissues of MIRI mice. miR-219a-2 overexpression decreased [Ca] and the expression of HIF1α and NR1 in hypoxia/reoxygenation (H/R)-treated HL-1 cells. Then, the luciferase reporter assay showed that miR-219a-2 inhibited the transcription of HIF1α and HIF1α promoted the transcription of NR1. Both HIF1α overexpression and NMDAR function enhancement removed the inhibitory effect of miR-219a-2 on calcium overload and cell apoptosis in H/R-treated HL-1 cells. Finally, the overexpression of miR-219a-2 decreased Ca concentration, cell apoptosis, and myocardial infarction size in MIRI mice, while the NMDAR function enhancer reversed the therapeutic effect of miR-219a-2.
MiR-219a-2 reducing NMDAR-mediated calcium overload via HIF1α/NR1 axis, thus alleviating cell apoptosis in MIRI.
在心肌缺血再灌注损伤（MIRI）过程中，细胞内钙浓度（[Ca]）持续增加，导致心肌细胞凋亡，最终导致心肌损伤，而钙超载的上游调控机制尚不清楚。本研究关注 miR-219a-2 在 MIRI 中的作用，并旨在阐述其对 MIRI 时钙超载的调节机制。
通过 qRT-PCR 测定 MIRI 小鼠心脏组织中 miR-219a-2 的表达。使用荧光微孔板读数器通过 fluo-3 测定[Ca]。通过 qRT-PCR 和 Western blot 测定低氧诱导因子 1α（HIF1α）和 N-甲基-D-天冬氨酸受体 1（NMDAR）必需亚基 1（NR1）的表达。使用荧光素酶报告基因检测法证实 miR-219a-2 与 HIF1α 之间的相互作用以及 HIF1α 与 NR1 之间的相互作用。通过 B 细胞淋巴瘤 2 相互作用介导体细胞死亡（Bim）的表达水平和 TUNEL 阳性细胞的数量来测定细胞凋亡。通过 TTC/Evans Blue 染色法测定小鼠心肌梗死面积。
miR-219a-2 在 MIRI 小鼠心脏组织中表达下调。miR-219a-2 过表达可降低缺氧/复氧（H/R）处理的 HL-1 细胞中的[Ca]和 HIF1α 和 NR1 的表达。然后，荧光素酶报告基因检测表明 miR-219a-2 抑制 HIF1α 的转录，而 HIF1α 促进 NR1 的转录。HIF1α 过表达和 NMDAR 功能增强消除了 miR-219a-2 在 H/R 处理的 HL-1 细胞中对钙超载和细胞凋亡的抑制作用。最后，miR-219a-2 的过表达降低了 MIRI 小鼠的 Ca 浓度、细胞凋亡和心肌梗死面积，而 NMDAR 功能增强剂逆转了 miR-219a-2 的治疗作用。
miR-219a-2 通过 HIF1α/NR1 轴减少 NMDAR 介导的钙超载，从而减轻 MIRI 中的细胞凋亡。
