DataSheet2_miR-3587 Inhibitor Attenuates Ferroptosis Following Renal Ischemia-Reperfusion Through HO-1.docx

Renal ischemia-reperfusion (IR) is frequently observed in patients who are critically ill, yet there are no reliable or effective approaches for the treatment of this condition. Ferroptosis, a form of programmed cell death, is regulated by key genes such as glutathione peroxidase 4 (GPX4) and heme oxygenase-1 (HMOX1) and participates in the injury of renal tubular epithelial cells during IR. This study aimed to investigate the miRNA-mRNA regulatory networks involved in ferroptosis following renal IR. Using bioinformatics analysis, HMOX1 was found to be significantly upregulated during the early stages of renal IR injury, and microRNA-3587 (miR-3587) was identified as a putative regulator of HMOX1. When a miR-3587 inhibitor was applied in a hypoxia-reoxygenation (HR) model system using renal tubular epithelial cells, HO-1 protein (encoded by HMOX1) expression was significantly increased relative to that observed in the HR group, with concomitant increases in GPX4 protein levels, enhanced cell viability, a reduction in malondialdehyde content, decreased Fe2+ level, and the restoration of normal mitochondrial membrane potential. Transmission electron microscopy showed a reduced or absent mitochondrial crest and a damaged mitochondrial outer membrane. Targeting of HMOX1 by miR-3587 was confirmed by luciferase reporter gene assay. In conclusion, these preliminary results indicate that inhibition of miR-3587 promotes HO-1 upregulation, thereby protecting renal tissues from IR-induced ferroptosis.

肾缺血再灌注（Renal ischemia-reperfusion, IR）在重症患者中较为常见，但目前尚无可靠有效的治疗手段。铁死亡（Ferroptosis）是一种程序性细胞死亡形式，受谷胱甘肽过氧化物酶4（glutathione peroxidase 4, GPX4）、血红素氧合酶-1（heme oxygenase-1, HMOX1）等关键基因调控，并参与缺血再灌注损伤过程中肾小管上皮细胞的损伤。本研究旨在探讨肾缺血再灌注后铁死亡相关的miRNA-mRNA调控网络。通过生物信息学分析，研究人员发现HMOX1在肾缺血再灌注损伤早期显著上调，并鉴定出microRNA-3587（miR-3587）作为HMOX1的潜在调控因子。在使用肾小管上皮细胞构建的缺氧复氧（hypoxia-reoxygenation, HR）模型中，施加miR-3587抑制剂后，由HMOX1编码的HO-1蛋白表达量较缺氧复氧组显著升高，同时GPX4蛋白水平也随之升高，细胞活力增强，丙二醛含量降低，Fe²+水平下降，线粒体膜电位恢复正常。透射电镜观察显示，线粒体嵴减少或消失，线粒体外膜受损。双荧光素酶报告基因实验（luciferase reporter gene assay）证实了miR-3587对HMOX1的靶向调控作用。综上，这些初步结果表明，抑制miR-3587可促进HO-1上调，从而保护肾组织免受缺血再灌注诱导的铁死亡损伤。