研究背景：急性心肌梗死（Acute myocardial infarction，AMI）作为急性冠脉综 合征的重要亚型，是以冠状动脉血供急剧减少或中断导致心肌持续性缺血坏死为特 征的危重心血管急症。临床治疗以再灌注治疗为核心策略，主要包括药物溶栓及机 械性再灌注手段，如经皮冠状动脉介入治疗（Percutaneous coronary intervention，PCI） 与冠状动脉旁路移植术（Coronary artery bypass grafting，CABG）。值得注意的是， 心肌组织在恢复血流灌注过程中可能引发缺血再灌注（Ischemia/Reperfusion，I/R） 损伤，这一矛盾现象表现为再灌注区域心肌细胞出现钙超载、氧自由基爆发及线粒 体功能障碍等特征性病理改变，进而导致心肌组织继发性损伤。因此，优化AMI患 者的诊断与及时干预对降低院内死亡率及改善远期预后具有重要临床价值。近年研 究表明，铁死亡（Ferroptosis）这一新型细胞死亡方式在心肌I/R损伤病理进程中发 挥关键调控作用。环状RNA（Circular RNA，circRNA）作为具有闭环结构的非编码 RNA（Non-coding RNA，ncRNA）分子，通过微小RNA（MicroRNA，miRNA）海 绵效应及蛋白互作等多重机制参与细胞铁稳态调节。 研究目的：本研究旨在探讨血清circPRDM5在AMI临床诊断中的应用价值， 深入研究circPRDM5与铁死亡之间的关系，并探讨其在调控心肌细胞铁死亡、减轻 心肌I/R损伤中的作用。通过阐明circPRDM5如何抑制铁死亡来减轻心肌I/R损伤 的机制，为AMI的诊断和治疗提供新的理论依据和潜在的治疗靶点。 研究方法：1.从基因表达综合（Gene Expression Omnibus，GEO）数据库中筛选 AMI中差异表达的关键circRNA；2.数据库中找到circPRDM5的详细信息，设计人 源（hsa_circ_0005654）和鼠源（mmu_circ_0001481）引物，通过实时荧光定量PCR （Quantitative real-time PCR，qRT-PCR）、琼脂糖凝胶电泳、Sanger 测序等方法验 证引物的特异性和正确性，通过放线菌素D、RNase R酶处理验证其环状结构和稳 定性；3.通过 qRT-PCR 检测血清 circPRDM5 的表达水平，通过受试者工作特征曲 线（Receiver operating characteristic curve，ROC）分析 circPRDM5 对 AMI 的临床诊 断价值；4.构建circPRDM5 的干扰载体和过表达载体并通过qRT-PCR 验证效率； 5.通过缺氧装置构建缺氧/复氧（Hypoxia/reoxygrnation，H/R）心肌细胞模型；6.通 过CCK-8法、乳酸脱氢酶（Lactate dehydrogenase，LDH）试剂盒、TUNEL染色等 方法分析心肌细胞的损伤程度；7.通过流式细胞仪、荧光显微镜、酶标仪等仪器检 测细胞中的活性氧自由基（Reactive oxygen species，ROS）、亚铁离子（Fe2+）、还 原型谷胱甘肽（Glutathione，r-glutamyl cysteingl +glycine，GSH）、丙二醛 （Malonaldehyde，MDA）水平来间接反映铁死亡程度；8.通过Western Blot实验研究circPRDM5 对铁死亡相关蛋白表达的影响；9.建立小鼠I/R模型，通过体内实验 验证circPRDM5 对铁死亡的调控作用，通过病理、生化检查和ELISA实验判断心 肌损伤程度；10.生物信息学预测及 RNA 结合蛋白免疫沉淀（RNA binding protein immunoprecipitation，RIP）验证 circPRDM5 与胚胎致死性异常视觉样蛋白1（ELAV Like RNA Binding Protein 1，ELAVL1）结合；11.构建 ELAVL1 的干扰和过表达载 体并通过 qRT-PCR 和 Western Blot 实验验证其干扰和过表达效率；12.通过 qRT PCR 和Western Blot 实验探索circPRDM5 和ELAVL1的互作模式；13.回复实验验 证ELAVL1逆转circPRDM5调控铁死亡对心肌损伤的影响。 研究结果：1.从 GEO 数据库中筛选出在 AMI 中表达下调的 circPRDM5； 2.CircPRDM5 的人源和鼠源引物设计正确，circPRDM5具有稳定的环状结构；3.血 清circPRDM5对AMI具有良好的诊断价值，ROC曲线下面积（Area under the curve， AUC）为 0.862[95% confidence interval（CI），0.814-0.909]，联合 cTnT 和肌酸激酶 同工酶（Creatine Kinase MB，CK-MB）可以提高诊断的灵敏度（96.6%）；4.血清 circPRDM5 的表达水平与Gensini 评分成负相关；5.心肌细胞中转染circPRDM5干 扰载体后其表达降低，感染circPRDM5过表达慢病毒载体后其表达升高；6.心肌I/R （心肌细胞 H/R）损伤中发生了铁死亡，过表达 circPRDM5 可以促进 GPX4 和 SLC7A11 的表达进而抑制心肌细胞铁死亡，提高心肌细胞存活率；7.过表达 circPRDM5 后心肌细胞中ROS、Fe2+、MDA、LDH水平均降低，GSH水平升高， 干扰circPRDM5 后出现相反的结果；8.体内实验发现 circPRDM5 对心肌具有保护 作用，过表达circPRDM5后小鼠心肌组织HE染色可见心肌损伤减轻，Western Blot 和免疫荧光结果可见GPX4和SLC7A11蛋白表达水平升高；9.与对照组相比，过表 达circPRDM5 后的小鼠左心室功能减退程度降低，此外，生化检查结果可见小鼠血 清CK-MB、LDH和cTnT水平均降低；10.ENCORI、RBPDB 和RPISeq 数据库中 筛选出ELAVL1并通过RIP实验验证ELAVL1与circPRDM5直接结合；11.在心肌 细胞中转染ELAVL1 干扰载体后其表达降低，转染ELAVL1过表达质粒后其表达 升高；12.心肌细胞中 circPRDM5 与 ELAVL1 相互抑制形成双向负反馈环； 13.ELAVL1 在 H/R 心肌细胞中表达上调；14.ELAVL1 可以促进心肌细胞铁死亡， 过表达ELAVL1后H/R心肌细胞存活率降低，细胞中ROS、Fe2+、MDA、LDH水 平均升高，GSH水平降低，干扰ELAVL1后出现相反的结果；15.回复实验结果可 见，加入过表达ELAVL1可以逆转过表达circPRDM5对心肌细胞铁死亡和损伤的 影响；16.回复实验中，加入干扰ELAVL1载体可以逆转干扰circPRDM5对心肌细 胞铁死亡和损伤的影响。 研究结论：血清circPRDM5可以作为AMI的诊断生物标志物；circPRDM5可 以作为AMI的保护因子抑制铁死亡减轻心肌I/R损伤；circPRDM5与ELAVL1形成双向负反馈环，circPRDM5 通过调节 ELAVL1 的表达抑制心肌细胞铁死亡从而 减轻心肌I/R损伤。

Background: Acute Myocardial Infarction (AMI), as an important subtype of acute coronary syndrome, is a critical cardiovascular emergency characterized by continuous ischemic necrosis due to a sharp reduction or interruption of coronary blood supply. Reperfusion therapy is the core strategy of clinical treatment, mainly including drug thrombolysis and mechanical reperfusion methods, such as Percutaneous coronary intervention. PCI and Coronary artery bypass grafting (CABG). It is noteworthy that myocardial tissue restoration of blood perfusion may lead to Ischemia/Reperfusion (I/R) injury, a contradiction manifested by characteristic pathological changes such as calcium overload, oxygen free radical outbreak, and mitochondrial dysfunction in myocardial cells in the reperfusion area, leading to secondary injury of myocardial tissue. Therefore, optimizing the diagnosis and timely intervention of AMI patients has important clinical value to reduce the in-hospital mortality and improve the long-term prognosis. Recent studies have shown that ferroptosis, a new cell death mode, plays a key regulatory role in the pathological process of myocardial I/R injury. circular RNA (circRNA), as a non coding RNA (ncRNA) molecule with closed-loop structure, is involved in cellular iron homeostasis regulation through multiple mechanisms such as microRNA (miRNA) sponge effect and protein interaction. Objectives: The purpose of this study was to investigate the application value of serum circPRDM5 in the clinical diagnosis of AMI, to further investigate the relationship between circPRDM5 and ferroptosis, and to explore its role in regulating ferroptosis of cardiomyocytes and reducing myocardial I/R injury. By elucidating the mechanism of how circPRDM5 inhibits ferroptosis and alleviates myocardial I/R injury, it provides a new theoretical basis and potential therapeutic target for the diagnosis and treatment of AMI. Methods: 1. Key circrnas differentially expressed in AMI were screened from Gene Expression Omnibus (GEO) database; 2. The detailed information of circPRDM5 was found in the database, and human primers (hsa_circ_0005654) and mouse primers (mmu_circ_0001481) were designed. quantitative real-time PCR was performed. The specificity and correctness of the primers were verified by qRT-PCR, agarose gel electrophoresis and Sanger sequencing. The ring structure and stability of the primers were verified by actinobacillin D and RNase R enzyme treatment. 3. The expression level of serum circPRDM5 was detected by qRT-PCR, and the clinical diagnostic value of circPRDM5 in AMI was analyzed by receiver operating characteristic curve (ROC). 4. The interference vector and overexpression vector of circPRDM5 were constructed and the efficiency was verified by qRT-PCR; 5. Hypoxia/reoxygrnation (H/R) cardiomyocyte model was constructed by hypoxia apparatus; 6. The degree of myocardial cell damage was analyzed by CCK-8, lactate dehydrogenase (LDH) kit and TUNEL staining. 7. Reactive oxygen species (ROS), ferrous ion (Fe2+), glutathione (glutathione) were detected by flow cytometry, fluorescence microscopy, enzyme labelling and other instruments. r glutamyl cysteingl +glycine, GSH) and Malonaldehyde (MDA) levels reflected the degree of ferroptosis indirectly. 8. The effect of circPRDM5 on the expression of ferroptosis related protein was studied by Western Blot. 9. Mouse I/R model was established to verify the regulatory effect of circPRDM5 on ferroptosis through in vivo experiments, and the degree of myocardial injury was determined by pathology, biochemical examination and ELISA test. 10. Bioinformatics prediction and RNA binding protein immunoprecipitation, RIP) verified the Binding of circPRDM5 to the embryonic lethal abnormal vision Like RNA binding Protein 1 (ELAV Like RNA Binding Protein 1, ELAVL1). 11. Construct the interference and overexpression vector of ELAVL1 and verify its efficiency; 12. The interaction pattern between circPRDM5 and ELAVL1 was explored by qRT-PCR and Western Blot experiments. 13. The response experiment verified that ELAVL1 reversed the effect of circPRDM5 on cardiac injury induced by ferroptosis. Results: 1. circPRDM5 with down-regulated expression in AMI was screened from GEO database; 2. The human and mouse primers of CircPRDM5 are correctly designed, and circPRDM5 has a stable ring structure; 3. Serum circPRDM5 has a good diagnostic value for AMI, and the area under the curve (AUC) was 0.862[95% confidence interval (CI), 0.814-0.909]. Combined cTnT and Creatine Kinase MB (CK-MB) could improve the diagnostic sensitivity (96.6%). 4. The expression level of serum circPRDM5 was negatively correlated with Gensini score. 5. The expression of cardiomyocytes decreased after transfection with circPRDM5 interfering vector, and increased after infection with circPRDM5 overexpressing lentiviral vector; 6. Ferroptosis occurs during myocardial I/R (cardiomyocyte H/R) injury. Overexpression of circPRDM5 can promote the expression of GPX4 and SLC7A11, thereby inhibiting ferroptosis of cardiomyocytes and improving the survival rate of cardiomyocytes. 7. The levels of ROS, Fe2+, MDA and LDH in cardiomyocytes decreased after overexpression of circPRDM5, while the levels of GSH increased, and the opposite results appeared after interference with circPRDM5. 8. In vivo experiments showed that circPRDM5 had a protective effect on myocardium. After overexpression of circPRDM5, the myocardium damage was alleviated by HE staining, and the protein expression levels of GPX4 and SLC7A11 were increased by Western Blot and immunofluorescence results. 9. Compared with the control group, the left ventricular function of mice overexpressed with circPRDM5 was reduced. In addition, the serum levels of CK-MB, LDH and cTnT were reduced by biochemical examination. 10. ELAVL1 was screened from ENCORI, RBPDB and RPISeq databases, and the direct combination of ELAVL1 and circPRDM5 was verified by RIP experiment; 11. The expression of ELAVL1 in cardiomyocytes was decreased after transfection with ELAVL1 interference vector, and increased after transfection with ElAVL1 overexpression plasmid; 12. circPRDM5 and ELAVL1 inhibit each other to form a bidirectional negative feedback loop in cardiomyocytes; 13. The expression of ELAVL1 was up-regulated in H/R cardiomyocytes. 14.ELAVL1 can promote ferroptosis of cardiomyocytes. After overexpression of ELAVL1, the survival rate of H/R cardiomyocytes is decreased, the levels of ROS, Fe2+, MDA and LDH in cells are increased, and the level of GSH is decreased. 15. According to the experimental results, adding ELAVL1 overexpression could reverse the effects of circPRDM5 overexpression on ferroptosis and injury of cardiomyocytes; 16. In the response experiment, the addition of interfering ELAVL1 vector could reverse the effects of interfering circPRDM5 on ferroptosis and injury of cardiomyocytes. Conclusions: Serum circPRDM5 can be used as a diagnostic biomarker of AMI. CircPRDM5 can be used as a protective factor of AMI to inhibit ferroptosis and reduce myocardial I/R injury. CircPRDM5 formed a bidirectional negative feedback loop with ELAVL1, and circPRDM5 inhibited ferroptosis of cardiomyocytes by regulating the expression of ELAVL1, thereby alleviating myocardial I/R injury.