IDDF2024-ABS-0285 Ferritinophagy activation and sideroflexin3-dependent mitochondrial iron overload contribute to biliary epithelial cells epithelial-mesenchymal transition and liver fibrosis in biliary atresia

<h3>Background</h3> Biliary atresia (BA) is a severe liver disease in neonates, which is caused by obliteration of the intra- and the extrahepatic biliary duct leading to cholestasis, and progressive liver injury and fibrosis. Epithelial-mesenchymal transformation (EMT) of bile duct epithelial cells is considered to be a key mechanism in the pathogenesis of liver fibrosis. The present study aimed to explore the role and underlying mechanism of ferroptosis in EMT and liver fibrosis. <h3>Methods</h3> Thirty-four cases of BA liver tissue and twenty-two cases of adjacent normal liver tissue from hepatoblastoma were subjected to RNA-sequencing and proteomic analysis to verify the expression differences of EMT and fibrosis between BA and non-BA liver tissues, as well as the correlation between the expression level of the differential gene SFXN3 in BA and clinical pathological characteristics. A mouse liver model of biliary atresia induced by rotavirus in rhesus monkeys was constructed to verify the expression differences of EMT and fibrosis-related genes. A fibrosis model was constructed by inducing cholangiocytes with TGF-β, and the effects of SFXN3 knockdown and overexpression on cellular ferroptosis, EMT, and fibrosis were studied. <h3>Results</h3> We found that EMT-related molecules were significantly upregulated in BA patients. In addition, RT-PCR and Western blot experiments on liver of BA mice also revealed significant activation of fibrosis and EMT-related molecules In terms of mechanism, we found that SFXN3 was significantly upregulated in the fibrotic liver of patients (IDDF2024-ABS-0285 Figure 1). And it is positively correlated with the degree of fibrosis, EMT and clinical assay index. We discovered through colocalization that SFXN3 is localized to the biliary epithelial cells (IDDF2024-ABS-0285 Figure 2). Mechanically, we found that nuclear receptor coactivator NCOA4, a master regulator of ferritin phagocytosis, significantly activates and degrades ferritin during EMT, thereby releasing large amounts of ferrous, further leading to SFXN3-dependent mitochondrial iron overload. Conversely, knockdown of NCOA4 or SFXN3 with small interfering RNAs could effectively ameliorate ferroptotic cell death, cellular or mitochondrial iron overload and lipid peroxides accumulation (IDDF2024-ABS-0285 Figure 3). <h3>Conclusions</h3> Overall, our findings underscore that ferritinophagy activation and SFXN3-dependent mitochondrial iron overload play critical roles in ferroptosis and EMT in biliary atresia.