miR-499在急性心肌梗死早期辅助诊断及治疗中的作用及其分子机制探讨

Myocardial infarction is a major cause of morbidity and mortality worldwide. The diagnosis of AMI is based on the "two of three" criteria, including typical chest symptoms, increase in the serum level of cardiac troponins T/I and typical ECG changes. Indeed, troponins T and I are sensitive and specific biomarkers of myocytes necrosis. However, it should be noted that it takes 2-4h for troponins to be released after the onset of AMI and the moderate elevation of troponins is common in chronic renal failure patients without significant myocardial damage. So, earlier biomarkers with both high sensitivity and specificity are urgently demanded. The diagnostic relevance of circulating microRNAs in AMI is a cutting-edge topic both for basic research and clinical implications. MiR-499 is a cardiac-abundant microRNA. We have previously found that miR-499 could protect neonatal rat cardiomyocytes against H2O2-induced apoptosis. We explore whether miR-499 could acts as an early diagnostic and monitoring biomarker; whether it possesses the property of the specific clinical classification and protects the cardiomyocytes. These works will be conducted in a rat AMI model and clinic AMI patients. Moreover, in AMI rats, we will prove that miR-499 could improve heart function by injecting recombinant miR-499 adenovirus into the caudal vein or borders of the infarct area and investigate whether miR-499 could be a protective biomarker. Finally, we will elucidate the molecular mechanisms that miR-499 protects cardiomyoctyes from apoptosis under pathological conditions. We have previously demonstrated that JNK signal rather than ERK and p38 is involved in the regulation of miR-499. Preliminary results also indicate that PACS2, PDCD4 and DYRK2 are the targets of miR-499. They co-participate to prevent the apoptosis of cardiomyocytes. Our final goal of this project is to construct a miR-499-centred the regulatory network in response to cardiac oxidative damage. The results will demonstrate the feasibility of miR-499 being the assisted diagnostic biomarker for AMI and provide the experimental basis for its applications in diagnosis and therapy of AMI in future.

寻找新的急性心肌梗死(AMI)特异而敏感的早期诊断方法及治疗手段，一直是医学工作者的奋斗目标。我们前期实验发现miR-499具有抑制细胞凋亡，保护心肌细胞的作用。本课题将通过对临床标本的检测和动物模型的建立，探讨miR-499是否可以作为AMI早期诊断及病程发展监控的生物标志；是否具有AMI临床分型特异性；miR-499是否对心肌细胞有保护作用；在AMI大鼠中，miR-499是否可以保护心脏功能，成为保护性标志物；阐明miR-499保护心肌细胞的分子机制，即JNK、ERK、p38等信号通路是否参与调节miR-499表达；构建在氧化应激损伤中，miR-499与其下游靶基因保护心肌细胞的调控网络。本课题的完成在一定程度上可明确miR-499作为AMI辅助诊断的生物标志物的可行性，为其在AMI诊断和治疗的潜在临床应用提供实验依据。

本课题研究显示急性心肌梗塞组miR-499表达量显著高于慢性心衰组，提示miR-499在一定程度上可以区分心肌细胞损伤的急性期和慢性期。对于急性心梗患者，miR-499的表达量在ST段抬高型患者和非ST抬高型患者中无显著差异，但是相比其他心肌酶学指标，miR-499表达时间更早，在胸痛发生1 h后即可被检出，持续时间更长，在胸痛发生24 h仍可被检出，因此对非ST段抬高型患者有更好的辅助诊断意义。.在细胞水平上，我们证实miR-499对氧化应激或者内毒素引起的心肌损伤都有保护作用。H2O2作用于心肌细胞，引起JNK信号通路激活，JNK和c-JUN发生磷酸化，p-c-JUN与AP-1形成复合体，促进Myh7b内含子中的miR-499转录，miR-499通过抑制下游靶基因Pdcd4和Pacs2，抑制BID转入线粒体及BID发生剪切形成t-BID，最终抑制线粒体凋亡途径，达到保护心肌细胞作用。此外，我们还证实miR-499对于内毒素引起的心肌细胞损伤同样具有保护作用。内毒素引起miR-499表达降低，miR-499通过抑制下游靶基因Pdcd4和Sox6而抑制细胞凋亡。.本课题另一个研究核心是揭示在心肌细胞分化晚期，抑制分化的Wnt和Notch信号分子被抑制和清除的分子机制。在心肌分化的后期，自噬水平增高，通过自噬作用降解beta catenin和NICD1，解除Wnt和Notch信号对心肌分化的抑制作用。mTOR信号通路在心肌分化后期活性降低，减少细胞核内beta catenin和NICD1蛋白量，同样抑制Wnt和Notch信号通路。Wnt通路抑制分子sFRP4在分化后期短暂增高，拮抗Wnt作用，但是sFRP4在心肌分化过程中不能持续表达，因为sFRP4可结合整合素受体alpha1beta1，使FAK磷酸化，活化的FAK与NICD1结合并进入细胞核，引起Notch1信号通路的激活，由此引起心肌分化的抑制。.