Identification of serum exosomal miRNA biomarkers in patients with lupus nephritis.

Abstract

Introduction: 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.

Methods: 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.

Results: 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.

Conclusions: 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.