Antrodia cinnamomea-derived extracellular vesicles suppress hepatocellular carcinoma progression by microRNA-mediated regulation of RAD51B-dependent DNA repair and PI3K/AKT signaling

Extracellular vesicles (EVs) derived from natural sources have emerged as biocompatible nanocarriers with therapeutic potential in cancer treatment. Antrodia cinnamomea , a medicinal fungus endemic to Taiwan, exhibits hepatoprotective and anticancer activities; however, the functional role and molecular mechanisms of its EVs in hepatocellular carcinoma (HCC) remain largely unexplored. EVs isolated from A. cinnamomea (AC-EVs) were characterized using size distribution analyses and electron microscopy. Their biological effects were examined in AML12 hepatocytes and multiple HCC cell lines through assays assessing cell growth, motility, invasion, apoptosis, and oxidative stress. Vesicle-associated microRNAs were profiled by small RNA sequencing, followed by functional validation using gain- and loss-of-function approaches to define downstream signaling events. AC-EVs were characterized as nanoscale, bilayered vesicles with favorable stability and biocompatibility. Functional assays demonstrated that AC-EVs suppressed proliferation, migration, and invasion of HCC cells while exhibiting lower cytotoxicity toward AML12 hepatocytes under the experimental conditions tested. Small RNA sequencing identified a novel vesicle-enriched miRNA, ac-EV-miR-5, which directly targeted RAD51B, a key regulator of homologous recombination-mediated DNA repair. Mechanistically, ac-EV-miR-5 was associated with reduced RAD51B expression, attenuation of PI3K/AKT signaling, and activation of mitochondrial apoptotic responses through modulation of BAX, BCL2, and Caspase-3. Notably, AC-EVs reduced ROS levels in AML12 hepatocytes while promoting ROS accumulation and oxidative stress-induced apoptosis in HCC cells. These findings demonstrate that AC-EVs function as natural nanovesicles carrying miRNA cargo associated with modulation of DNA repair and survival signaling in liver cancer cells, highlighting fungal EV-based miRNA delivery as a promising therapeutic strategy for hepatocellular carcinoma.