Identification and validation of bile exosomal microRNA signatures for diagnosing acute rejection in liver transplant recipients.

Introduction: Acute rejection (AR) remains a major complication affecting graft function and recipient prognosis after liver transplantation (LT). Bile exosomal microRNAs (miRNAs) possess organ specificity, local enrichment, and high stability, making them promising non-invasive liquid biopsy biomarkers for the early detection of AR. Methods: In this study, bile samples were collected from 30 LT recipients with AR and 30 recipients with stable graft function (non-AR). Exosomes were isolated via ultracentrifugation and characterized. Total RNA was extracted and subjected to small RNA sequencing, followed by bioinformatics analysis. Candidate differentially expressed miRNAs were then validated using Real-Time Quantitative PCR (qRT-PCR) in an expanded cohort. Results: Sequencing revealed 63 significantly upregulated and 4 downregulated miRNAs in the AR group compared with the non-AR group. Target genes of candidate differentially expressed miRNAs were enriched in key immune-regulatory pathways, including PI3K-Akt and MAPK signaling. qRT-PCR validation further confirmed that the expression levels of miR-181a-5p, miR-200c-3p, and miR-192-5p in bile exosomes were significantly higher in the AR group (P < 0.0001). Discussion: Our findings identify and validate bile exosomal miRNA signatures for AR in LT recipients, supporting these miRNAs as a low-risk, organ-specific liquid biopsy strategy for AR diagnosis, with potential for clinical translation in post-transplant monitoring.