Extracellular vesicles derived microRNAs as non-invasive markers of liver fibrosis in chronically infected HCV patients: a pilot study

Extracellular vesicles (EVs) are an increasingly promising tool for liquid biopsy in liver diseases. Hepatitis C Virus (HCV) infection, alone or together with Human Immunodeficiency Virus (HIV) infection significantly impacts on the microRNA (miRNA) EVs content resembling chronic hepatitis C (CHC) progression. The objective of the study was to delve into the intricate EVs-miRNA profiles in CHC patients with different liver fibrosis stages, aiming to pinpoint non-invasive markers capable of distinguishing significant fibrosis. Plasma EV-miRNAs from 50 CHC patients (HCV+ and HCV+/HIV+) stratified in no significant (F < 2) and significant (F ≥ 2) fibrosis, were massively sequenced. General linear models (GLM) were used to identify significantly differential expressed (SDE) miRNAs according to liver fibrosis stages (F ≥ 2 and F < 2). Dysregulated biological pathways were subsequently analyzed <i>in silico</i> for the following groups: i) all patients; ii) HCV+; and iii) HCV+/HIV+. Multiple-ordered logistic regression analysis was performed to develop a score to identify F ≥ 2 cases. The diagnostic potential of both the SDE miRNAs and the developed score was assessed using ROC curve analysis. With respect to all CHC patients, two SDE miRNAs (hsa-miR-122-5p and hsa-miR-92a-3p) were identified which regulate genes related to cytoskeleton organization. Regarding their diagnostic performance to discriminate F ≥ 2, both miRNAs individually demonstrated acceptable diagnostic values. However, their combined use in a new score enhanced their diagnostic performance (AUROC = 0.833). In the HCV+ subgroup, 8 SDE miRNAs (hsa-miR-122-5p, hsa-miR-320c, hsa-miR-3615, hsa-miR-320a-3p, hsa-miR-374b-5p, hsa-let-7a-3p, hsa-miR-199a-5p, hsa-miR-142-5p), which regulate macrophage activity and cell growth/death regulation, were recognized. Among them, hsa-miR-3615 displayed the highest diagnostic performance to discriminate F ≥ 2 (AUROC = 0.936). With respect to HCV+/HIV+, 18 SDE miRNAs (hsa-miR-4508, hsa-miR-122-5p, hsa-miR-451a, hsa-miR-1290, hsa-miR-1246, hsa-miR-107, hsa-miR-15b-5p, hsa-miR-194-5p, hsa-miR-22-5p, hsa-miR-20b-5p, hsa-miR-142-5p, hsa-miR-328-3p, hsa-miR-335-3p, hsa-miR-125a-5p, hsa-miR-423-3p, hsa-let-7d-3p, hsa-miR-128-3p, hsa-miR-10a-5p) were recognized that regulate RNA silencing processes. In this case, hsa-miR-423-3p and hsa-miR-128-3p showed outstanding diagnostic performances (AUROC > 0.900). Distinct EVs-miRNA profiles were identified in patients with varying liver fibrosis stages, both in the overall CHC cohort and within HCV+ and HCV+/HIV+ subgroups. These specific miRNA signatures would allow the elucidation of potential mechanisms involved in clinical evolution and identification of specific biomarkers of unfavorable progression, plausible to be used in a diagnostic panel. Furthermore, the developed score demonstrates the ability to discriminate within the CHC group those individuals with significant fibrosis regardless of their HIV infection status.