Exosomal microRNAs (exo-miRs) have been promising cancer biomarkers. MiRs in hepatocellular carcinoma (HCC) cell-derived exosomes (HEX) were analyzed to identify reliable serum biomarkers for HCC. To detect overexpressed miRs in HEX, extracted exosomal small RNAs from human HCC cell lines and normal hepatocytes were sequenced and analyzed. Clinical significance of the overexpressed miRs in HEX was evaluated using quantitative real-time PCR (qRT-PCR) on serum samples of a validation cohort consisting of 28 healthy individuals, 60 with chronic liver disease, and 90 with HCC. We found 49 significantly overexpressed miRs in HEX compared to a normal hepatocyte. Among them, miR-10b-5p, miR-18a-5p, miR-215-5p, and miR-940 were overexpressed in HCC tissues and also associated with prognosis of HCC in the analysis of a public omics database. qRT-PCR analysis of the four serum exo-miRs in the validation cohort revealed serum exo-miR-10b-5p as a promising biomarker for early-stage HCC with 0.934 area under the curve (AUC) (sensitivity, 90.7%; specificity, 75.0%; cutoff value, 1.8-fold). Overexpression of serum exo-miR-215-5p was found to be significantly associated with poor disease-free survival in patients with HCC. Serum exo-miR-10b-5p is a potential biomarker for early-stage HCC, while serum exo-miR-215-5p can be used as prognostic biomarker for HCC.

This study aimed to identify novel long noncoding RNA (lncRNA) biomarkers for hepatocellular carcinoma (HCC) using publicly available tissue genomic datasets and validate their diagnostic utility for early‐stage HCC. Differentially expressed lncRNAs between 371 HCC and 50 nontumor tissues were obtained from The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA_LIHC) project. Subsequently, the expression of the serum‐ and extracellular vesicle (EV)‐derived lncRNA was assessed in 10 patients with HCC and 10 healthy controls using RT–qPCR. The candidate lncRNAs were validated in 90 HCC and 92 non‐HCC (29 healthy control, 28 chronic hepatitis, 35 liver cirrhosis) patients. The sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) were calculated for the candidate lncRNAs and the current HCC biomarker, alpha‐fetoprotein (AFP). SFTA1P, HOTTIP, HAGLROS, LINC01419, HAGLR, CRNDE, and LINC00853 were markedly upregulated in HCC in TCGA_LIHC dataset. Among them, LINC00853 has not been reported in relation to HCC before. In patients with HCC, only expression of small EV‐derived LINC00853 (EV‐LINC00853) was increased. EV‐LINC00853 showed excellent discriminatory ability in the diagnosis of all‐stage HCC (AUC = 0.934, 95% confidence interval = 0.887–0.966). Moreover, using a 14‐fold increase and 20 ng·mL⁻¹ as cutoffs for EV‐LINC00853 expression and AFP level, respectively, EV‐LINC00853 was found to have a sensitivity of 93.75% and specificity of 89.77%, while AFP showed only 9.38% sensitivity and 72.73% specificity for the diagnosis of early‐stage HCC (mUICC stage I). EV‐LINC00853 had a positivity of 97% and 67% in AFP‐negative and AFP‐positive early HCC, respectively. Serum EV‐derived LINC00853 may be a novel potential diagnostic biomarker for early HCC, especially for AFP‐negative HCC.

Hepatocellular carcinoma (HCC) has a high rate of cancer recurrence (up to 70%) in patients who undergo surgical resection. We investigated prognostic gene signatures for predicting HCC recurrence using in silico gene expression analysis. Recurrence-associated gene candidates were chosen by a comparative analysis of gene expression profiles from two independent whole-transcriptome datasets in patients with HCC who underwent surgical resection. Five promising candidate genes, CETN2, HMGA1, MPZL1, RACGAP1, and SNRPB were identified, and the expression of these genes was evaluated using quantitative reverse transcription PCR in the validation set (n = 57). The genes CETN2, HMGA1, RACGAP1, and SNRPB, but not MPZL1, were upregulated in patients with recurrent HCC. In addition, the combination of HMGA1 and MPZL1 demonstrated the best area under the curve (0.807, 95% confidence interval [CI] = 0.681–0.899) for predicting HCC recurrence. In terms of clinicopathological correlation, CETN2, MPZL1, RACGAP1, and SNRPB were upregulated in patients with microvascular invasion, and the expression of MPZL1 and SNRPB was increased in proportion to the Edmonson tumor differentiation grade. Additionally, overexpression of CETN2, HMGA1, and RACGAP1 correlated with poor overall survival (OS) and disease-free survival (DFS) in the validation set. Finally, Cox regression analysis showed that the expression of serum alpha-fetoprotein and RACGAP1 significantly affected OS, whereas platelet count, microvascular invasion, and HMGA1 expression significantly affected DFS. In conclusion, HMGA1 and RACGAP1 may be potential prognostic biomarkers for predicting the recurrence of HCC after surgical resection.

This study investigated the diagnostic potential of serum small extracellular vesicle‐derived long noncoding RNAs (EV‐lncRNAs) for hepatocellular carcinoma (HCC). Driver oncogenic lncRNA candidates were selected by a comparative analysis of lncRNA expression profiles from two whole transcriptome human HCC datasets (Catholic_LIHC and TCGA_LIHC). Expression of selected lncRNAs in serum and small EVs was evaluated using quantitative reverse transcription PCR. Diagnostic power of serum EV‐lncRNAs for HCC was determined in the test (n = 44) and validation (n = 139) cohorts. Of the six promising driver onco‐lncRNAs, DLEU2, HOTTIP, MALAT1, and SNHG1 exhibited favorable performance in the test cohort. In the validation cohort, serum EV‐MALAT1 displayed excellent discriminant ability, while EV‐DLEU2, EV‐HOTTIP, and EV‐SNHG1 showed good discriminant ability between HCC and non‐HCC. Furthermore, a panel combining EV‐MALAT1 and EV‐SNHG1 achieved the best area under the curve (AUC; 0.899, 95% CI = 0.816–0.982) for very early HCC, whereas a panel with EV‐DLEU2 and alpha‐fetoprotein exhibited the best positivity (96%) in very early HCC. Serum small EV‐MALAT1, EV‐DLEU2, EV‐HOTTIP, and EV‐SNHG1 may represent promising diagnostic markers for very early‐stage HCC.

Exosomal microRNAs (exo-miRs) contribute to cancer metastasis. To identify pro-metastatic circulating exo-miRs in hepatocellular carcinoma (HCC), next-generation sequencing-based plasma exo-miR profiles of 14 patients with HCC (eight non-metastatic and six with metastasis within 1 year of follow-up) were analyzed. Sixty-one miRs were significantly overexpressed among patients with metastatic HCC. Candidate miRs were selected through integrative analyses of two different public expression datasets, GSE67140 and The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA_LIHC). Integrative analyses revealed 3 of 61 miRs (miR-106b-5p, miR-1307-5p, and miR-340-5p) commonly overexpressed both in metastasis and vascular invasion groups, with prognostic implications. Validation was performed using stored blood samples of 150 patients with HCC. Validation analysis showed that circulating exo-miR-1307-5p was significantly overexpressed in the metastasis group (p = 0.04), as well as in the vascular invasion and tumor recurrence groups. Circulating exo-miR-1307-5p expression was significantly correlated with tumor stage progression (p < 0.0001). Downstream signaling pathways of miR-1307 were predicted using TargetScan and Ingenuity Pathway Analysis. On comprehensive bioinformatics analysis, the downstream pathway of miR-1307-5p, promoting epithelial–mesenchymal transition (EMT), showed SEC14L2 and ENG downregulation. Our results show that circulating exo-miR-1307-5p promotes metastasis and helps predict metastasis in HCC, and SEC14L2 and ENG are target tumor suppressor genes of miR-1307 that promote EMT.

Protein markers of hepatocellular carcinoma (HCC)-derived exosomes (HEX) have not yet been fully evaluated. Here, we identified novel protein contents of HEX and their clinical significance as biomarkers. Exosomes were isolated from human HCC cell lines and an immortalized normal hepatocyte cell line. Proteomic analyses revealed 15 markedly overexpressed proteins in HEX. The clinical relevance of the 15 proteins was analyzed in public RNA-sequencing datasets, and 6 proteins were selected as candidate of potential biomarkers. Serum CCT8 and CFL1 were markedly overexpressed in test cohort (n = 8). In the validation cohort (n = 224), the area under the curve (AUC) of serum CCT8 and CFL1 for HCC diagnosis was calculated as 0.698 and 0.677, respectively, whereas that of serum alpha-fetoprotein (AFP) was 0.628. The combination of three serum markers (CCT8, CFL1, and AFP) demonstrated the highest AUC for HCC diagnosis. (AUC = 0.838, 95% confidence interval = 0.773–0.876) Furthermore, higher serum CCT8 and CFL1 concentrations were significantly associated with the presence of vascular invasion, advanced tumor stage, poor disease-free survival, and poor overall survival. Cofilin-1 and CCT8, enriched proteins in HEX, were identified as potential diagnostic and prognostic serum biomarkers for HCC patients.