miR-197-3p在川崎病冠状动脉内皮细胞损伤中的功能及其作用机制研究

Kawasaki Disease (KD) is characterized by activation of immune system and general lesion of vascular endothelial system, especially the coronary artery lesion. It has become one of the major cause of children acquired heart disease. Yet the etiology and pathogenesis of KD is unknown, our previous study found that several miRNA may jointly serve as specific diagnostic biomarker of Kawasaki disease. Among them, we further found that miR-197-3p have significant effects on the viability and cell cycle of human coronary artery endothelial cells (HCAEC). This miRNA is highly up-regulated and highly secreted in HCAEC when stimulated by KD serum. Therefore, we hypothesized that this miRNA plays an important role in lesion of the coronary artery endothelial cells of Kawasaki Disease. We aim to identify miR-197-3p target genes and clarify the regulatory mechanism by manipulating the expression of this miRNA in HCAEC in the context of KD. This will provide insights on the mechanistic effect of miR-197-3p in coronary artery endothelial cells of KD, offering theoretical basis for the prevention and treatment of KD-derived coronary artery lesion.

川崎病(KD)以免疫系统活化和血管内皮系统广泛损害为特征，尤以冠状动脉损伤最为严重，成为儿童后天获得性心脏病的主要原因之一，然而，KD的病因及致病机制至今不明。我们前期研究发现几种miRNA联合起来可能作为KD特异性诊断标志物，从中进一步筛选，发现miR-197-3p对人冠状动脉内皮细胞（HCAEC）活性和周期有显著影响，在KD患儿血清刺激下该miRNA在HCAEC中显著上调，同时也被大量分泌。因此，我们推测该miRNA在KD冠状动脉内皮细胞损伤中发挥重要功能及调控机制。因此，在此工作基础上，本课题拟通过在KD患儿血清作用下在HCAEC中对miR-197-3p表达进行干预,筛选和确定miR-197-3p调控的靶基因，探索miR-197-3p在类似KD环境下对HCAEC功能的影响及其调控机制，阐明miR-197-3p对KD冠状动脉内皮细胞调控的分子机制，为KD致冠状动脉损伤防治提供理论依据。

目的：川崎病（Kawasaki disease，KD）发病率逐年上升, 其导致冠脉损伤的机制还未研究清楚。我们以前的研究发现异常的microRNA与KD冠脉损伤之间有很强的关联性。本课题研究了miR-197-3p/TIMP3在KD诱导冠脉损伤中的作用及其机制。.方法与结果：在KD病人血清中miR-197-3p水平显著升高。同时，KD患者血清培养的HCAECs（human coronary artery endothelial cells）中的miR-197-3p水平也明显上调。转染miR-197-3p mimic可降低HCAECs的细胞活力，并且打乱HCAECs细胞周期。通过iTRAQ标记和质谱技术对转染miR-197-3p mimic组进行了定量蛋白组学分析。结果表明，有328个蛋白被miR-197-3p mimic调节。利用IPA软件对这些差异蛋白进行网络分析，同时结合双荧光素酶报告实验，我们发现TIMP3、ANGPT2及MCM3可能是miR-197-3p的靶基因。并进一步证实了TIMP3是miR-197-3p靶基因。临床样本和细胞中TIMP3及内皮损伤相关蛋白THBS1、VWF及HSPG2检测显示, TIMP3水平与内皮损伤相关蛋白THBS1、VWF及HSPG2表达呈负相关。通过动物实验也验证了这一结果，进一步研究,发现，HSPG2可下调TIMP3及TIMP3诱导的HCAECs损伤。.总结：KD可诱导miR-197-3p在病人血清及其刺激的HCAECs中的表达, 下调靶基因TIMP3的表达，导致人冠状动脉内皮损伤。冠状动脉内皮损伤所导致的HSPG2上调可进一步抑制TIMP3的表达。因此，本项目研究揭示了miR-197-3p/TIMP3水平的异常可能是KD诱导冠状动脉内皮损伤的重要机制，为临床KD治疗提供了新的靶点。.