The Exosome Encapsulated microRNAs as Circulating Diagnostic Marker for Hepatocellular Carcinoma with Low Alpha fetoprotein.

: Diagnosis of Hepatocellular carcinoma (HCC) remains challenging to clinicians, particularly in a patient with low Alpha fetoprotein. Here, in-silico, ex-vivo, and in-vitro data were combined to identify liver-specific exosomal miRNAs as an early diagnostic marker for HCC. Transcriptome profiling for mRNA and small RNA in same HCV-HCC and normal liver tissues followed by cross-validation of 41 deregulated miRNAs (log2 FoldChange>1.5, padj <0.1) with GEO/TCGA datasets of HCV/HBV related HCC vs. normal/adjacent tissue revealed three miRNAs were commonly deregulated (miR-10b/miR-21/miR-182) among all HCC irrespective of viral etiology. Targets of top deregulated miRNAs were identified by TargetScan/miRwalk and validated in mRNA transcriptome data followed by Panther/Gene Ontology enrichment/Cytoscape analysis suggested that targets were mostly from carcinogenesis pathways. Hence, those miRNAs were validated in normal and HCV-HCC tissues by qRT-PCR and subsequently in plasma-derived-exosomes of both HBV/HCV infected non-HCC [chronic hepatitis (CH)/liver cirrhosis (LC)] and HCC samples, and in liver-specific Anti-Asgr2 immuno-enriched exosomes. Exosomes were verified using Nanosight/TEM/immune-blotting with anti-Alix/anti-GRP78/anti-Asgr2. Along with miR-21-5p, miR-10b-5p/miR-221-3p/miR-223-3p was found significantly upregulated in the exosome of HCC patients than CH/non-HCC. The comparable expression pattern was seen in anti-Asgr2 immuno-precipitated exosomes. Interestingly, the AFP level was found below 250ng/ml in about 94% of HCV-HCC and 62% of HBV-HCC patients. ROC analysis showed that miR-10b-5p+miR-221-3p+miR-223-3p+miR-21-5p could differentiate CH/non-HCC(CH+LC) from HCC with AUROC: 0.86[97.5% CI (0.77-0.94)]/0.80[97.5% CI (0.70-0.89)], sensitivity:74%/58% and specificity:86%/95% while miR-10b-5p+miR-221-3p+miR-223-3p showed better AUROC: 0.84[97.5% CI (0.74-0.94)/0.74[(97.5% CI (0.63-0.84)], sensitivity: 86%/86% and specificity:66%/53% for low AFP-HCC vs. CH/non-HCC respectively than the combination of four miRNAs. Multivariate analysis further revealed low Albumin and high miR-21-5p as probable independent risk factor for HCC.