ABSTRACT Hepatocellular carcinoma (HCC) has a poor prognosis and high mortality. Ferroptosis, an iron‐dependent regulated cell death process, is implicated in cancer development and treatment. Wnt signaling and lysyl oxidase (Lox) family members are associated with ferroptosis. This study investigates how Wnt3a and/or Loxl2 knockdown affects liver cancer stem cells (LCSCs) and orthotopic tumor growth in mice, and explores the role of ferroptosis‐related genes. Bioinformatics identified ferroptosis‐ and HCC‐associated differentially expressed genes (DEGs) correlated with Wnt3a/Loxl2. LCSCs sorted from Hep3B were transduced with lentivirus for gene knockdown. Ferroptosis markers and DEG expression were analyzed. Wnt3a/Loxl2 knockout mice were generated using CRISPR‐Cas9, and orthotopic tumor models were established. Tumor inhibition rates, ferroptosis‐related indicators, and DEG expression were assessed. 199 ferroptosis‐related DEGs were identified in HCC; ZEB1 was selected as a key gene via PPI analysis. Wnt3a/Loxl2 knockdown increased Fe 2+ and MDA, and decreased GSH, most evidently in double‐knockdown cells. In vivo, single‐ and double‐knockout groups showed suppressed tumor growth, with inhibition rates of 51%, 71%, and 93%, respectively. Tumor tissues exhibited similar ferroptosis marker changes. ZEB1 was upregulated in both cellular and animal knockout models. Wnt3a/Loxl2 knockdown promotes ferroptosis in LCSCs and inhibits orthotopic tumor growth, with the strongest effect following dual‐gene knockout. ZEB1 may be an important regulatory factor in this process.
Ren et al. (Mon,) studied this question.