Data Sheet 1_Serum miRNA-186-3P and miRNA-382-3P constitute a novel Diagnostic miRNA signature for palindromic rheumatism.docx

BackgroundPalindromic rheumatism (PR) is a unique disease characterized by the intermittent inflammation of different joints that may progress to a variety of immune-related diseases. Unclear diagnostic criteria have limited the research on its pathogenesis and treatment options. Recently, microRNAs (miRNAs) have been used in the diagnosis of various diseases; however, the role of miRNAs in PR diagnosis remains unexplored. Using next-generation high-throughput sequencing (NGS), this study aimed to screen miRNAs specifically expressed in the serum of patients with PR to construct a miRNA signature and verify its diagnostic efficacy.
MethodsPatients with PR (N=4), patients with rheumatoid arthritis (RA; N=3), and healthy individuals (Con; N=3) were included in an exploration cohort. Differentially expressed miRNAs were screened using NGS to construct a miRNA signature, and bioinformatics tools were used to perform target gene enrichment analysis of the top 25 differentially expressed miRNAs, both upregulated and downregulated. RT-qPCR was used to verify the differential expression of the miRNA signature in three validation cohorts of patients with PR (N=27) and RA (N=30), and healthy individuals (N=31). The efficiency of the miRNA signature was evaluated using receiver operator characteristic (ROC) curves, an analytical method that assesses diagnostic accuracy.
ResultsA total of 130 miRNAs were differentially expressed in the PR exploration cohort, including 35 upregulated and 95 downregulated compared to levels in the RA and healthy cohorts. miRNA-186-3p showed the largest upregulated difference and miRNA-382-3p the largest downregulated difference; these were selected to construct the miRNA signature. In the ROC curve of the validation cohort, the PR miRNA signature produced an area under the ROC curve (AUC) of 0.980 (95% CI 0.942–1.000) when distinguishing from healthy individuals and of 0.906 (95% CI 0.830–0.983) when distinguishing from RA patients. However, miRNA-186-3p and miRNA-382-3p levels were not associated with disease activity in patients with PR.
ConclusionA miRNA signature comprising miRNA-186-3p and miRNA-382-3p can effectively diagnose and differentiate PR from RA. This study provides a basis for the creation of a clinical miRNA signature for the diagnosis of PR.

背景：回纹型风湿症（Palindromic Rheumatism, PR）是一类独特的疾病，以多关节间歇性炎症为核心特征，病情可进展为多种免疫相关疾病。目前其诊断标准尚未明确，这极大限制了发病机制与治疗方案相关研究的开展。近年来，微小核糖核酸（microRNAs, miRNAs）已被广泛应用于多种疾病的诊断，但miRNAs在PR诊断中的作用仍未得到探索。本研究借助下一代高通量测序（next-generation high-throughput sequencing, NGS）技术，旨在筛选PR患者血清中特异性表达的miRNAs，以构建miRNA诊断特征标签，并验证其诊断效能。
方法：本研究纳入PR患者（N=4）、类风湿关节炎（rheumatoid arthritis, RA）患者（N=3）及健康对照个体（Con, N=3）组成探索队列。通过NGS筛选差异表达miRNAs以构建miRNA诊断特征标签，并采用生物信息学工具对上调及下调排名前25的差异表达miRNAs开展靶基因富集分析。本研究设置3个验证队列，采用实时定量逆转录聚合酶链式反应（RT-qPCR）验证该miRNA诊断特征标签的差异表达情况，验证队列共纳入PR患者27例、RA患者30例及健康对照个体31例。采用受试者工作特征（receiver operator characteristic, ROC）曲线——一种评估诊断准确性的经典分析方法——评估该miRNA诊断特征标签的诊断效能。
结果：在PR探索队列中，共筛选得到130个差异表达miRNAs，其中相较于RA队列与健康对照队列，上调表达35个，下调表达95个。miRNA-186-3p的上调差异幅度最大，miRNA-382-3p的下调差异幅度最大，二者被选中用于构建miRNA诊断特征标签。在验证队列的ROC曲线分析中，该PR相关miRNA诊断特征标签区分PR患者与健康对照个体的ROC曲线下面积（area under the ROC curve, AUC）为0.980（95%置信区间：0.942~1.000），区分PR患者与RA患者的AUC为0.906（95%置信区间：0.830~0.983）。但miRNA-186-3p与miRNA-382-3p的表达水平与PR患者的疾病活动度无显著相关性。
结论：由miRNA-186-3p与miRNA-382-3p组成的miRNA诊断特征标签可有效诊断PR，并实现PR与RA的精准区分。本研究为构建用于PR临床诊断的miRNA诊断特征标签提供了重要的理论依据。