Analysis of plasma extracellular vesicle-derived microRNAs from patients with nonalcoholic fatty liver disease and hepatocellular carcinoma

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 plasma EV-miRNA profiles between NBNC-HCC and control groups including non-alcoholic fatty liver disease (NAFLD) and healthy controls using NanoString method. The differentially expressed EV-miRNAs were validated in another set of plasma samples by qRT-PCR. Five 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. Among them, EV-miR-19-3p exhibited the best diagnostic performance and displayed a high sensitivity for detecting AFP-negative HCC and early-stage HCC, indicating EV-miR-19-3p could serve as a novel circulating biomarker for the diagnosis and prognosis of NBNC-HCC. To mechanistically describe NAFLD, we demonstrated the role of EVs in mediating communication between macrophages and lipotoxic hepatocytes. Macrophages were transfected with a Cy3-miR-223 mimic and co-cultured with lipotoxic hepatocytes. We found that miR-223 was highly expressed in EV fractions from the transfected macrophages, as opposed to the protein fractions. Upon co-culture, the lipotoxic hepatocytes displayed Cy3 fluorescence and exhibited an increase in miR-223 levels and a decrease in miR-223 target genes, FOXO3 and TAZ, as compared to the control group. Blocking EV secretion from macrophages with GW4869 led to reduced transfer of miR-223 to hepatocyte recipient cells. Furthermore, the MemGlow dye was transferred to lipotoxic hepatocytes when incubated with MemGlow-labeled EVs from macrophages. The results also suggested that low-density lipoprotein receptor (LDLR) played a partial role in facilitating EV uptake by lipotoxic hepatocytes. In summary, our findings suggest that macrophages can transfer transfected miRNA to lipotoxic hepatocytes.