Diagnostic exosomal microRNAs and pathogenic regulatory networks in hepatocellular carcinoma revealed by exosomal RNA sequencing: A case–control study

Objective Hepatocellular carcinoma is a leading cause of cancer mortality, and early detection remains limited by the low sensitivity of alpha-fetoprotein. This study aimed to identify serum-derived exosomal microRNAs with diagnostic potential for hepatocellular carcinoma and to explore their regulatory molecular networks. Methods This observational case–control study included 50 patients with hepatocellular carcinoma and 50 matched healthy controls. Serum exosomes were isolated and verified by transmission electron microscopy, nanoparticle tracking analysis, and flow cytometry for CD9, CD63, and CD81. High-throughput small RNA sequencing (three hepatocellular carcinoma samples vs three control samples) identified differentially expressed microRNAs. Predicted target genes were analyzed using protein–protein interaction networks based on the Search Tool for the Retrieval of Interacting Genes/Proteins database, Gene Ontology analysis, and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Candidate microRNAs were validated by reverse transcription quantitative polymerase chain reaction, and diagnostic performance was evaluated using receiver operating characteristic analysis. Results Seven differentially expressed microRNAs were identified, and bioinformatic network analysis revealed forkhead box O1 and serine/arginine-rich splicing factor 11 as central hub genes potentially targeted by hsa-miR-27a-3p and hsa-miR-493-3p, respectively. Reverse transcription quantitative polymerase chain reaction confirmed significant upregulation of miR-27a-3p ( P  =   0.003) and downregulation of miR-493-3p ( P  =   0.014) in hepatocellular carcinoma. Receiver operating characteristic analysis demonstrated high diagnostic accuracy for miR-493-3p (area under the curve = 0.840) and miR-27a-3p (area under the curve = 0.827). Importantly, the combined biomarker panel (alpha-fetoprotein, miR-27a-3p, and miR-493-3p) achieved markedly improved performance (area under the curve = 0.943), outperforming alpha-fetoprotein alone (area under the curve = 0.828). Conclusions Serum exosomal miR-27a-3p and miR-493-3p exhibit strong diagnostic potential and are associated with forkhead box O1 and serine/arginine-rich splicing factor 11. These findings highlight their promise as complementary biomarkers and provide new insight into exosome-mediated molecular regulation in hepatocarcinogenesis.