Objectives: To analyze the differentially expressed exosomal miRNAs in patients with chronic heart failure (CHF) complicated by hyperuricemia (HUA) and explore their potential as novel diagnostic molecular markers and their target genes.
Methods: This study was conducted among 30 CHF patients with HUA (observation group) and 30 healthy volunteers (control group) enrolled between September, 2020 and September, 2023. Peripheral blood samples were collected from 6 CHF patients with HUA for analyzing exosomal miRNAs by high-throughput sequencing, and the results were validated in the remaining 24 patients using qRT-PCR. GO and KEGG enrichment analyses were performed to predict the the target genes of the identified differential miRNAs. We also validated the differentially expressed miRNAs by animal experiment.
Results: A total of 42 differentially expressed exosomal miRNAs were detected in observation group by high-throughput sequencing; among them, miR-27a-5p was significantly upregulated (P=0.000179), and miR-139-3p was significantly downregulated (P=0.000058). In the 24 patients with both CHF and PUA, qRT-PCR validated significant upregulation of miR-27a-5p (P=0.004) and downregulation of miR-139-3p (P=0.005) in serum exosomes. When combined, miR-27a-5p and miR-139-3p had a maximum area under the curve (AUC) of 0.899 (95% CI: 0812-0.987) for predicting CHF complicated by HUA. GO and KEGG enrichment analyses suggested that the differential expressions of miR-27a-5p and miR-139-3p was associated with the activation of the AMPK-mTOR signaling pathway to activate the autophagic response. We obtained the same conclusion from animal experiment.
Conclusions: Upregulated exosomal miR-27a-5p combined with downregulated exosomal miR-139-3p expression can serve as a novel molecular marker for diagnosis of CHF complicated by HUA, and their differential expression may promote autophagy in cardiomyocytes by activating the AMPK-mTOR signaling pathway.
目的: 分析慢性心力衰竭（CHF）合并高尿酸血症（HUA）患者血清外泌体miRNA的差异表达，探讨其作为CHF合并HUA新型诊断分子标志物的可能性，并对差异miRNA进行靶基因功能分析，分析作用靶点。方法: 以2020年9月~2023年9月南京中医药大学附属南京中医院心血管病科收治的CHF合并HUA患者为观察组（n=30），选择同期健康志愿者为对照组（n=30）。两组各选取6例样本，采用高通量测序分析血清外泌体中的差异表达miRNA，采用RT-PCR检测对未作高通量测序的观察组和对照组样本（n=24）进行验证，使用R软件进行GO、KEGG富集分析，预测差异表达miRNA的作用靶点，并通过动物实验验证临床筛查的差异miRNA。结果: 高通量测序分析显示，观察组患者共检测到42个差异表达的miRNA（18个上调，24个下调），其中miR-27a-5p上调（P<0.001），miR-139-3p下调（P<0.001）。RT-PCR检测显示，观察组患者血清外泌体中miR-27a-5p表达量上调（P=0.004）、miR-139-3p表达量下调（P=0.005）；ROC曲线下面积（AUC）分析发现，miR-27a-5p、miR-139-3p预测CHF合并HUA发病的AUC分别是0.708（95% CI：0.562-0.855）和0.734（95% CI：0.593-0.876），两者联合预测CHF与HUA发病的AUC为0.899（95% CI：0.812-0.987）。对差异基因进行GO富集分析发现，细胞自噬是富集程度最高的靶点；KEGG功能注释显示，激活AMPK-mTOR信号通路可能是差异表达的miR-27a-5p和miR-139-3p作用靶点之一。进一步动物实验得到了相同的验证。结论: 血清外泌体中miR-27a-5p上调和miR-139-3p下调可作为精准诊断CHF合并HUA的新型分子标志物，激活AMPK-mTOR信号通路后促进心肌细胞的自噬反应可能是差异表达的miR-27a-5p、miR-139-3p的作用靶点之一。.
Keywords: AMP-activated protein kinases; TOR serine-threonine kinases; autophagy; chronic heart failure; exosomes; hyperuricemia; microRNAs.