背景:2型糖尿病(T2DM)和冠状动脉疾病(CAD)通常是并存的临床实体,在全球范围内发病率仍在增长。最近,循环microRNAs(miRNAs)已经成为心脏代谢疾病的新型分子参与者。本研究旨在确定特定的miRNA特征作为T2DM中CAD的候选生物标志物,并描绘促成糖尿病动脉粥样硬化的潜在miRNA依赖性机制。
方法:总共38个来自有或没有CAD的T2DM患者的血浆样本,使用微阵列收集CAD患者和健康对照用于2,578个miRNA的表达谱。探讨差异表达(DE)-miRNA靶基因的调控作用,利用多种生物信息学工具进行功能注释和途径富集分析。然后,利用Cytoscape软件中的STRING数据库建立蛋白质-蛋白质相互作用网络,其次是聚类分析和枢纽基因鉴定。在94名参与者的较大复制队列中进行逆转录定量实时聚合酶链反应(RT-qPCR)用于微阵列数据验证。应用接受者操作特征分析来评估miRNA的诊断价值。使用多变量逻辑回归分析来建立基于miRNA的诊断模型。
结果:在发现阶段,hsa-miR-4505,hsa-miR-4743-5p,hsa-miR-6846-5p,和下调hsa-miR-3613-3p,hsa-miR-4668-5p,hsa-miR-4706,hsa-miR-6511b-5p,hsa-miR-6750-5p,hsa-miR-4750-3p,hsa-miR-320e,hsa-miR-4717-3p,在T2DM-CAD患者中检测到hsa-miR-7850-5p。DE-miRNA靶基因显著富集钙离子结合,肌动蛋白细胞骨架的调节,和基因表达。hsa-miR-4505,hsa-miR-4743-5p,和hsa-miR-4750-3p被发现参与脂肪酸代谢,白细胞跨内皮迁移,和神经营养蛋白信号通路。hsa-miR-4505,hsa-miR-4743-5p,与T2DM受试者和对照组相比,T2DM-CAD患者中的hsa-miR-4750-3p(均p<0.001)通过RT-qPCR进一步证实。所有验证的miRNA对T2DM-CAD表现出良好的鉴别值(AUC=0.833-0.876)。3种miRNA的组合在T2DM中检测CAD的最佳性能(AUC=0.959,100%灵敏度,86.67%特异性)。
结论:我们的研究揭示了T2DM合并CAD患者血浆来源的miRNA的独特特征。还鉴定了潜在的miRNA调节途径,探讨T2DM中CAD的潜在发病机制。我们开发了hsa-miR-4505,hsa-miR-4743-5p和hsa-miR-4750-3p的特异性三miRNA组,这可能是T2DM患者CAD的一种新型非侵入性生物标志物。
BACKGROUND: Type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) are commonly coexisting clinical entities with still growing incidence worldwide. Recently, circulating microRNAs (miRNAs) have emerged as novel molecular players in cardiometabolic diseases. This
study aimed to identify a specific
miRNA signature as a candidate biomarker for CAD in T2DM and to delineate potential
miRNA-dependent mechanisms contributing to diabetic atherosclerosis.
METHODS: A total of 38 plasma samples from T2DM patients with and without CAD, CAD patients and healthy controls were collected for expression profiling of 2,578 miRNAs using microarrays. To investigate the regulatory role of differentially expressed (DE)-miRNA target genes, functional annotation and pathway enrichment analyses were performed utilizing multiple bioinformatics tools. Then, protein-protein interaction networks were established leveraging the STRING database in Cytoscape software, followed by cluster analysis and hub gene identification. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was carried out for microarray data validation in the larger replication cohort of 94 participants. Receiver operating characteristic analysis was applied to evaluate the diagnostic values of miRNAs. Multivariate logistic regression analysis was used to develop
miRNA-based diagnostic models.
RESULTS: In the discovery stage, overexpression of hsa-miR-4505, hsa-miR-4743-5p, hsa-miR-6846-5p, and down-regulation of hsa-miR-3613-3p, hsa-miR-4668-5p, hsa-miR-4706, hsa-miR-6511b-5p, hsa-miR-6750-5p, hsa-miR-4750-3p, hsa-miR-320e, hsa-miR-4717-3p, hsa-miR-7850-5p were detected in T2DM-CAD patients. The DE-miRNA target genes were significantly enriched in calcium ion binding, regulation of actin cytoskeleton, and gene expression. hsa-miR-4505, hsa-miR-4743-5p, and hsa-miR-4750-3p were found to be involved in fatty acid metabolism, leukocyte transendothelial migration, and neurotrophin signaling pathway. Dysregulation of hsa-miR-4505, hsa-miR-4743-5p, and hsa-miR-4750-3p in T2DM-CAD patients compared with T2DM subjects and controls (all p < 0.001) was further confirmed by RT-qPCR. All validated miRNAs demonstrated good discriminatory values for T2DM-CAD (AUC = 0.833-0.876). The best performance in detecting CAD in T2DM was achieved for a combination of three miRNAs (AUC = 0.959, 100% sensitivity, 86.67% specificity).
CONCLUSIONS: Our
study revealed a unique profile of plasma-derived miRNAs in T2DM patients with CAD. Potential
miRNA-regulated pathways were also identified, exploring the underlying pathogenesis of CAD in T2DM. We developed a specific three-miRNA panel of hsa-miR-4505, hsa-miR-4743-5p and hsa-miR-4750-3p, that could serve as a novel non-invasive biomarker for CAD in patients with T2DM.