Hepatocyte-derived extracellular vesicles are enriched in miR-122-5p that sensitizes hepatic stellate cells to ferroptosis

Liver fibrosis is a common feature of hepatitis and advanced steatotic liver diseases, but FDA-approved anti-fibrotic drugs are very limited. We previously found that extracellular vesicles (EVs) from normal hepatocytes have therapeutic actions in models of liver fibrosis in mice. MicroRNA (miR)-122-5p is mainly produced by hepatocytes, constitutes 70% of total hepatic miRNAs, and regulates cholesterol and iron homeostasis. Here we show that miR-122-5p is the principal miR in EVs from mouse hepatocytes and induces ferroptosis in HSC, a novel mechanism which likely contributes to anti-fibrotic actions of hepatocyte EVs. Mouse AML12 hepatocyte EVs were isolated by differential ultracentrifugation, AML12 EVs ameliorated liver necrotic lesions and fibrogenesis induced by short-term CCl 4 injury. Small RNA-seq was performed to profile AML12 EV miRNA components. Of the 156 miRNAs identified, miR-122-5p was the most abundant. EVs from AML12 cells exacerbated erastin-induced ferroptosis in the human hepatic stellate cell (HSC) line LX2. Overexpression of miR-122-5p in LX2 cells led to dramatic enhancement of ferroptosis induced by erastin, RSL-3, FIN56, or cystine depletion. miR-122-5p-induced cell death was reversed by inhibitors of ferroptosis but not of apoptosis, necroptosis, or pyroptosis. Intracellular ferrous iron was significantly elevated by miR-122-5p. Wildtype or a 3’ GU-rich motif mutant but not a 5’ seed sequence mutant miR-122-5p mimic maintained the ability to promote ferroptosis. HSC sensitivity to miR-122-5p-mediated ferroptosis was greater in HSC exhibiting a higher level of activation. miR-122-5p-overexpressing LX2 cells demonstrated suppressed levels of GPX4 and of NRF1, a reported regulator of GPX4. Stable knockdown in LX2 cells of GPX4 or NRF1, but not in cells treated with scramble controls, led to enhanced ferroptosis, whereas overexpression of NRF1 partially reversed the susceptibility. 3’UTR reporter assay confirmed the regulation of NRF1 by miR-122-5p. In a human liver organoid (HLO) fibrogenesis model, hepatocyte EVs or miR-122-5p lentiviral delivery efficiently reduced fibrogenesis and induced ferroptosis, with the hepatocyte EV effect being reversed by a ferroptosis inhibitor. Anti-fibrogenic actions of hepatocyte EVs occur in part by potentiating HSC ferroptosis through delivery of miR-122-5p, which leads to elevated cellular ferrous iron levels and reduction of GPX4 expression by directly targeting NRF1.