Steatotic hepatocyte-derived extracellular vesicle miR-192 modulates M2-associated marker expression by suppressing IL6ST

Introduction Extracellular vesicles, which transport microRNAs, are emerging biomarkers for metabolic dysfunction-associated steatotic liver disease. Although extracellular vesicles modulate nearby hepatocytes, their influence on macrophages – central to disease progression – remains unclear. We aimed to identify functional microRNAs in extracellular vesicles and elucidate their role in macrophage activation and polarization. Material and Methods Extracellular vesicles were isolated from steatotic AML12 hepatocytes using ultracentrifugation and characterized by transmission electron microscopy. Cellular uptake was confirmed using PKH67 labeling and confocal microscopy. RAW264.7 macrophages were treated with extracellular vesicles or microRNA mimics. Predicted microRNA targets were validated using a dual luciferase assay and quantitative real-time polymerase chain reaction (PCR). Macrophage polarization markers (M1/M2) were analyzed using quantitative real-time PCR. Statistical significance was set at p < 0.05 (GraphPad Prism). Results Extracellular vesicles from steatotic AML12 hepatocytes showed enrichment of micro­RNAs (e.g., mmu-miR-122, -21, and -34a), with mmu-miR-192 showing the greatest increase (2.2-fold; p < 0.01). These extracellular vesicles were taken up by macrophages. Treatment with extracellular vesicle-derived miR-192 suppressed IL6ST expression (p < 0.05) and inhibited expression of M1 markers (e.g., iNOS, TNF-a, and IL-1b), while enhancing the expression of M2 markers (e.g., CD206 and IL-10) (p < 0.05). Restoring IL6ST expression reversed these effects. Conclusions Our findings suggest that extracellular vesicles may mediate miR-192 transfer from steatotic hepatocytes to macrophages in metabolic dysfunction-associated steatotic liver disease. By targeting IL6ST and shifting macrophage marker expression toward an M2-associated profile, the miR-192/IL6ST axis may contribute to the regulation of steatotic liver inflammation and warrants further investigation as a potential therapeutic target.