The effect of USP17 knockdown on autophagic ferroptosis in gastric cancer by regulating the BNIP3-NCOA4-FTH1 axis.

Gastric cancer (GC) is one of the most crucial malignancies worldwide because of the high lethality. This study aims to explore the mechanism by which ubiquitin-specific protease 17 (USP17) mediates autophagic ferroptosis in GC. In the present study, we cultured human GC cell line AGS in vitro and knocked BNIP3 or NCOA4 down using specific small interfering-RNA. Erastin was chosen as a ferroptosis inducer while Ferrostatin-1 was utilized as an inhibitor of ferroptosis. Then, the levels of mitophagy-related proteins, USP17, BNIP3, mitochondrial marker proteins, NCOA4, and FTH1 were quantified through RT-qPCR and Western blot tests. Also, the JC-1 method was adopted to detect mitochondrial membrane potential. Additionally, CCK-8 cell viability test was performed. Ultimately, glutathione, reactive oxygen species, malondialdehyde, and active Fe2+ were determined as indicators related to ferroptosis using corresponding kits. The interaction between USP17 and BNIP3 was assessed through co-immunoprecipitation. Genetic inhibition of BNIP3 reduced mitophagy and increased ferroptosis in GC cells. Subsequently, upon BNIP3 silencing, we found up-regulated NCOA4 and down-regulated FTH1, with autophagy inhibition resulting in the similar changes in the NCOA4–FTH1 pathway and cellular Fe2+ levels. NCOA4 inhibition partly counteracted BNIP3 deficiency-induced ferroptosis, indicating that BNIP3-dependent autophagic ferroptosis was associated with the NCOA4–FTH1 pathway. Furthermore, it was found that USP17 stabilized BNIP3 by reducing its ubiquitination level, which was linked to the regulatory role of USP17 in ferroptosis. USP17 knockdown modulates autophagic ferroptosis in GC by affecting the stability of BNIP3 and mediating the NCOA4–FTH1 pathway.