Chen Fei, Gao Jia, Shi Bo, Liu Wenjing, Gong Jianmin, Khan Adeel, Sun Yifan, Yang Ping, Li Zhiyang
Department of Clinical Laboratory, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, China.
Department of Orthopedics, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Clin Rheumatol. 2025 May 3. doi: 10.1007/s10067-025-07447-3.
Characterized by glomerulonephritis, lupus nephritis (LN) worsens the prognosis of patients with systemic lupus erythematosus (SLE) and contributes to its increased mortality rate. Here, we investigated whether serum exosomal microRNAs (miRNAs) are promising new biomarkers of LN.
Serum exosomes were initially isolated using a reagent-based kit, and total RNA was extracted with the Trizol method. Small RNA sequencing was subsequently performed to identify differentially expressed exosomal miRNAs. Validation of these miRNAs was conducted via real-time quantitative polymerase chain reaction (RT-qPCR) on individual samples from both the training and validation cohorts, leading to the identification of candidate small RNAs specifically associated with LN. Receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic performance of the identified candidates. To further investigate the immune landscape, 12 types of cytokines were quantified using flow cytometry, and their correlations with the candidate miRNAs were analyzed via Spearman rank correlation. Additionally, the biological functions of these miRNAs were explored through enrichment analyses based on Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
The levels of hsa-miR-497-5p and hsa-miR-6515-5p were significantly higher in the LN group compared to the SLE without LN group, with a combined area under the ROC curve (AUC) of 0.798. Notably, these two miRNAs demonstrated exceptional discriminative performance in identifying LN patients with mild proteinuria, achieving an AUC of 0.844. Furthermore, flow cytometry analysis revealed markedly elevated serum levels of interferon (IFN)-γ, interleukin (IL)-8, and IL-17 in the LN group compared to healthy controls (HCs). Additionally, IL-6 and IL-17 levels were significantly higher in the LN group compared to the SLE without LN group. Hsa-miR-6515-5p exhibited a strong positive correlation with IL-8 and IFN-γ. Bioinformatic analyses indicated that these exosomal miRNAs may contribute to the progression of LN by regulating key signaling pathways, with the MAPK signaling pathway being prominently implicated.
Our study demonstrated that serum exosomal miRNAs, specifically hsa-miR-497-5p and hsa-miR-6515-5p, show significant potential as biomarkers for the early detection and prognosis of LN.
狼疮性肾炎（LN）以肾小球肾炎为特征，会使系统性红斑狼疮（SLE）患者的预后恶化，并导致其死亡率上升。在此，我们研究了血清外泌体微小RNA（miRNA）是否有望成为LN的新型生物标志物。
首先使用基于试剂的试剂盒分离血清外泌体，然后采用Trizol法提取总RNA。随后进行小RNA测序以鉴定差异表达的外泌体miRNA。通过实时定量聚合酶链反应（RT-qPCR）对来自训练队列和验证队列的个体样本进行这些miRNA的验证，从而鉴定出与LN特异性相关的候选小RNA。采用受试者工作特征（ROC）曲线分析来评估所鉴定候选物的诊断性能。为了进一步研究免疫格局，使用流式细胞术对12种细胞因子进行定量，并通过Spearman等级相关性分析它们与候选miRNA的相关性。此外，基于基因本体论（GO）术语和京都基因与基因组百科全书（KEGG）通路，通过富集分析探索这些miRNA的生物学功能。
与无LN的SLE组相比，LN组中hsa-miR-497-5p和hsa-miR-6515-5p的水平显著更高，ROC曲线下面积（AUC）合并值为0.798。值得注意的是，这两种miRNA在识别轻度蛋白尿的LN患者方面表现出卓越的鉴别性能，AUC为0.844。此外，流式细胞术分析显示，与健康对照（HC）相比，LN组中干扰素（IFN）-γ、白细胞介素（IL）-8和IL-17的血清水平显著升高。此外，与无LN的SLE组相比，LN组中IL-6和IL-17水平显著更高。Hsa-miR-6515-5p与IL-8和IFN-γ呈强正相关。生物信息学分析表明，这些外泌体miRNA可能通过调节关键信号通路促进LN的进展，其中MAPK信号通路显著相关。
我们的研究表明，血清外泌体miRNA，特别是hsa-miR-497-5p和hsa-miR-6515-5p，作为LN早期检测和预后的生物标志物具有显著潜力。
