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. 2023 Nov 7;24(22):16043.
doi: 10.3390/ijms242216043.

Circulating Extracellular Vesicle-Derived microRNAs as Novel Diagnostic and Prognostic Biomarkers for Non-Viral-Related Hepatocellular Carcinoma

Bootsakorn Boonkaew  1 , Nantawat Satthawiwat  1 , Nutcha Pinjaroen  2 , Natthaya Chuaypen  1 , Pisit Tangkijvanich  1
Affiliations

Circulating Extracellular Vesicle-Derived microRNAs as Novel Diagnostic and Prognostic Biomarkers for Non-Viral-Related Hepatocellular Carcinoma

Bootsakorn Boonkaew et al. Int J Mol Sci. .

Abstract

Extracellular vesicle-derived microRNAs (EV-miRNAs) are promising circulating biomarkers for chronic liver disease. In this study, we explored the potential significance of plasma EV-miRNAs in non-hepatitis B-, non-hepatitis C-related HCC (NBNC-HCC). We compared, using the NanoString method, plasma EV-miRNA profiles between NBNC-HCC and control groups including patients with non-alcoholic fatty liver disease (NAFLD) and healthy controls. The differentially expressed EV-miRNAs were validated in another set of plasma samples by qRT-PCR. A total of 66 significantly differentially expressed EV-miRNAs between the HCC and the control groups were identified in the discovery set. In the validation cohort, including plasma samples of 70 NBNC-HCC patients, 70 NAFLD patients, and 35 healthy controls, 5 plasma EV-miRNAs were significantly elevated in HCC, which included miR-19-3p, miR-16-5p, miR-223-3p, miR-30d-5p, and miR-451a. These miRNAs were found to participate in several cancer-related signaling pathways based on bioinformatic analysis. Among them, EV-miR-19-3p exhibited the best diagnostic performance and displayed a high sensitivity for detecting alpha-fetoprotein-negative HCC and early-stage HCC. In multivariate analysis, a high EV-miR-19-3p level was demonstrated as an independently unfavorable predictor of overall survival in patients with NBNC-HCC. In conclusion, our data have indicated, for the first time, that EV-miR-19-3p could serve as a novel circulating biomarker for the diagnosis and prognosis of NBNC-HCC.

Keywords: biomarker; extracellular vesicles; hepatocellular carcinoma; microRNAs; nonalcoholic fatty liver disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic diagram of the study protocol and characterization of EVs derived from plasma. (a) Diagram of plasma EV isolation protocol, (b) Representative nanoparticle tracking plot for size distribution from a sample, (c) Quantification of particle size diameter (nm), and (d) Concentration (particles/mL) of plasma EVs from the healthy control, NAFLD, and NBNC-HCC groups. (e) Expression of EV markers, CD63, HSP70, and TSG101, and hepatocyte-specific receptor, ASGPR1 by Western blotting. (f) Transmission electron microscopy images of EVs from a sample. Scale bars, 200 μm. Data are presented as means ± S.E.M, ** p < 0.01.
Figure 2
Figure 2
Transcriptome profiling of miRNAs from plasma EVs using NanoString microarray. (a) Volcano plot of all differentially expressed miRNAs in the NBNC-HCC samples compared with the NAFLD samples, and (b) the NBNC-HCC samples compared with healthy controls. The significantly up-regulated and down-regulated miRNAs are denoted as red and blue dots, respectively.
Figure 3
Figure 3
Gene Ontology (GO) analysis of the differentially up-regulated EV miRNAs. Top 10 significantly enriched GO terms of biological process, molecular function, and KEGG pathways (p < 0.05).
Figure 4
Figure 4
Validation of candidate miRNAs in plasma EV using qRT-PCR. The relative expressions of the following: (a) miR-19-3p, (b) miR-16-5p, (c) miR-223-3p, (d) miR-30d-5p, and (e) miR-451a, in plasma EVs of healthy controls (n = 35), patients with NAFLD (n = 70), and patients with NBNC-HCC (n = 70). Data are presented as mean ± S.E.M., normalized with a reference miRNA, miR-3144-3p, and expressed relative to those of healthy controls. * p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 5
Figure 5
Receiver operating characteristic (ROC) curves of the candidate miRNAs for distinguishing between NBNC-HCC and non-HCC. (a) The area under the curve (AUC), and (b) the cut-off value and the discriminatory performance of each EV-miRNAs.
Figure 6
Figure 6
Kaplan–Meier survival curves for overall survival analysis of patients with NBNC-HCC (a) plasma EV-miR-19-3p, and (b) plasma EV-miR-16-5p.
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