hnRNPK regulates Wnt/β-catenin signaling pathway to inhibit ferroptosis in breast cancer

Background and purpose: Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA special binding protein that participates in regulating the expression of related genes and protein translation. It has been linked to the malignant occurrence and development of various tumors, but its role in breast cancer remains unclear. The aim of this study was to investigate the effects of hnRNPK on ferroptosis in breast cancer cells and the underlying mechanisms. Methods: Based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, hnRNPK expression in breast cancer tissues and normal tissues and its relationship with clinical prognosis were analyzed by bioinformatics. We detected hnRNPK expression in breast cancer cells and tissues using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemistry staining diagnosis methods. MCF-7 and MDA-MB-231 breast cancer cells were transfected with siRNA, and divided into control group (control), empty body group (NC), and interference vector group (si-hnRNPK). Cell proliferation was detected by cell counting kit-8 (CCK-8) and plate clone formation assays. RNA-seq analysis was applied to explore potential targeted biological functions and signaling pathways affected by hnRNPK. Additionally, we investigated the impact of hnRNPK on ferroptosis phenotype using Western blot and commercial kits for reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and Fe2+, ferroptosis inhibitor (ferrostatin-1, Fer-1) was used to detect the rescue effect of hnRNPK knockdown on ferroptosis. The impact of hnRNPK on the expressions of Wnt/β-catenin pathway-related proteins were determined by Western blot. Results: The bioinformatics analyses indicated hnRNPK was upregulated in breast cancer tissues (P<0.01), and the overall survival of patients in the high expression group was poorer compared with those in the low expression group (P<0.05). hnRNPK was highly expressed in breast cancer tissues and cells, and knocking down hnRNPK weakened the proliferation ability of breast cancer cells (P<0.05). The RNA-seq analysis showed that hnRNPK was significantly enriched in ferroptosis, apoptosis, and the Wnt/β-catenin signaling pathway. Knocking down hnRNPK promoted ferroptosis in breast cancer cells by inducing lipid ROS and MDA, as well as Fe2+ accumulation (P<0.05). Interestingly, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the promotive effect of hnRNPK knockdown on ferroptosis (P<0.05). Downregulation of hnRNPK led to a decrease in the expressions of β-catenin and c-Myc in the Wnt/β-catenin signaling pathway, while expressions of CK1α, APC and the GSK-3β complex were elevated (P<0.05). Conclusion: hnRNPK is highly expressed in breast cancer, and knocking down hnRNPK promotes ferroptosis in breast cancer cells by inhibiting the Wnt/β-catenin signaling pathway, thereby suppressing the malignant progression of breast cancer.