ABSTRACT

The present study aimed to assess the role of miR-1275 in cardiac ischemia reperfusion injury. H9 human embryonic stem cell (hESC)-derived cardiomyocytes stimulated by oxygen-glucose deprivation/reoxygenation (OGD/R) were used to simulate myocardial injury in vitro. miR-1275 expression levels in cells were measured by RT-qPCR. The release of lactate dehydrogenase (LDH) and creatine kinase (CK) was examined through LDH and CK ELISA kits. Cell apoptosis was detected through flow cytometry. A Fura-2 Calcium Flux Assay Kit and a Fluo-4 assay kit were used to determine the Ca²⁺ concentration. Expression levels of proteins were tested by Western blotting. The binding effect of miR-1275 and neuromedin U type 1 receptor (NMUR1) was detected by dual-luciferase activity assay. The results showed that miR-1275 was upregulated in OGD/R-stimulated cardiomyocytes. Inhibition of miR-1275 suppressed the increased activity of LDH and CK, cell apoptosis, reactive oxygen species (ROS) production, intracellular Ca²⁺ concentration and sarcoplasmic reticulum (SR) Ca²⁺ leak induced by OGD/R treatment in cardiomyocytes. miR-1275 directly targets 3ʹUTR of NMUR1 and negatively regulates NMUR1 expression. Silence of NMUR1 abolished the protecting effect of the miR-1275 antagomir on myocardial OGD/R injury. Our study indicated that the miR-1275 antagomir protects cardiomyocytes from OGD/R injury through the promotion of NMUR1.

摘要

本研究旨在评估 miR-1275 在心脏缺血再灌注损伤中的作用。H9 人胚胎干细胞 (hESC) 衍生的心肌细胞由氧-葡萄糖剥夺/复氧 (OGD/R) 刺激，用于模拟体外心肌损伤。通过 RT-qPCR 测量细胞中 miR-1275 的表达水平。通过 LDH 和 CK ELISA 试剂盒检测乳酸脱氢酶 (LDH) 和肌酸激酶 (CK) 的释放。通过流式细胞术检测细胞凋亡。Fura-2 钙流检测试剂盒和 Fluo-4 检测试剂盒用于测定 Ca ²⁺专注。通过蛋白质印迹测试蛋白质的表达水平。双荧光素酶活性测定法检测miR-1275与神经调节蛋白U型1受体（NMUR1）的结合作用。结果显示 miR-1275 在 OGD/R 刺激的心肌细胞中上调。抑制 miR-1275 可抑制 LDH 和 CK 活性增加、细胞凋亡、活性氧 (ROS) 产生、细胞内 Ca ²⁺浓度和肌浆网 (SR) Ca ²⁺心肌细胞中OGD/R处理引起的渗漏。miR-1275 直接靶向 NMUR1 的 3ʹUTR 并负调节 NMUR1 表达。NMUR1 的沉默消除了 miR-1275 antagomir 对心肌 OGD/R 损伤的保护作用。我们的研究表明，miR-1275 antagomir 通过促进 NMUR1 保护心肌细胞免受 OGD/R 损伤。