MRPL35 Suppresses Autophagy-Mediated Ferroptosis in Hepatocellular Carcinoma via the AMPK/mTOR Axis.
INTRODUCTION Ferroptosis and autophagy are recently identified forms of non-apoptotic regulated cell death with strong therapeutic implications for hepatocellular carcinoma (HCC). Mitochondrial ribosomal protein L35 (MRPL35), a mitochondria- specific component of mitoribosomes, has been proposed as a potential therapeutic target for liver cancer. The study focused on autophagy-dependent ferroptosis in HCC, with an emphasis on the functional significance of MRPL35. METHODS Bioinformatics approaches, RT-qPCR, along with western blotting, were used to determine MRPL35 expression in HCC. CCK-8, scratch, Matrigel, and flow cytometric assays were used to detect cell proliferation, migration, invasion, and apoptosis. JC-1 staining was used to detect mitochondrial function. Assay kits and C11-BODIPY assay were used to detect GSH, GSSG, MDA, ROS, Fe2+, ATP, and lipid ROS levels. MDA staining and western blotting assessed the presence of autophagosomes and autophagy-related proteins. In addition, western blotting was used to detect γ-H2AX, apoptosis-, ferroptosis, and AMPK/mTOR axis-related proteins. The in vivo effects of MRPL35 were confirmed by subcutaneous tumorigenesis assays in nude mice. RESULTS MRPL35 showed abnormally high expression in HCC with a low survival rate. MRPL35 downregulation suppressed cell viability and metastasis, promoted cell apoptosis and autophagy, as well as ROS-dependent DNA damage, inhibited tumor growth, and promoted ferroptosis and autophagy in HCC-bearing mice. MRPL35 interference also promoted autophagy-mediated ferroptosis. In addition, MRPL35 modulated the AMPK/mTOR axis, and AMPK inhibitor Compound C or AMPKα interference reversed the effects of MRPL35 interference on autophagy-mediated ferroptosis. DISCUSSION The elucidation of MRPL35's role in mediating the AMPK/mTOR axis is consistent with the well-established pro-tumorigenic role of MRPL35 and the significance of the AMPK/mTOR axis in HCC. The newly characterized MRPL35- AMPK/mTOR axis not only proposes MRPL35 as a therapeutic target for HCC but also advances our understanding of its carcinogenic mechanisms. CONCLUSION Overall, a novel regulatory mode of autophagy-reliant ferroptosis was established by the MRPL35/AMPK/mTOR axis in HCC.