RRM2 Promotes the Progression of Intrahepatic Cholangiocarcinoma by Inhibiting Ferroptosis Through the HIF ‐1α Pathway

ABSTRACT Background Intrahepatic cholangiocarcinoma (ICC) is a malignant tumour associated with a poor prognosis. Recent studies have suggested that ribonucleotide reductase subunit M2 (RRM2) could promote tumour progression. This study aims to explore the expression, function and mechanism of RRM2 in ICC by regulating ferroptosis through the HIF‐1α signalling pathway. Methods The expression of RRM2 and HIF‐1α was detected in ICC patients, and the relationship between RRM2 and prognosis was analysed. CCK‐8, clone formation assay, wound healing assay, transwell invasion experiment, cell apoptosis detection and tumorsphere formation assay were conducted to evaluate the effect of RRM2 on the progression of ICC. Transcriptomics was used to explore the mechanism of RRM2 promotion of ICC. Ferroptosis inducer (Ferrostatin‐1) and inducer (RSL3), HIF‐1α inhibitor (LW6) and inducer (CoCl 2 ) were applied to investigate the effects of RRM2 on reactive oxygen species (ROS), ferroptosis‐related indicators and HIF‐1α expression. The effects of RRM2 on tumour growth were validated using subcutaneous tumours in nude mice. Results RRM2 was significantly upregulated in ICC tissues and was associated with a poor prognosis in patients. Overexpression of RRM2 promoted the proliferation, colony formation, invasion, metastasis, stemness, and inhibited apoptosis of ICC cells. Transcriptomics clarified that the HIF‐1α signalling pathway was a key downstream pathway. The expression of HIF‐1α was significantly positively correlated with the expression of RRM2 and was associated with poor prognosis in patients. Overexpression of RRM2 upregulated HIF‐1α and reduced ROS levels. RRM2 upregulated SLC7A11 and GPX4 and downregulated ACSL4 by regulating the HIF‐1α signalling pathway, inhibiting intracellular iron ion accumulation and lipid peroxidation, thus suppressing ferroptosis. Under hypoxic conditions, the anti‐tumour effect of knocking down RRM2 was weakened. HIF‐1α inhibitor could reverse the ferroptosis inhibition caused by RRM2 overexpression, and the HIF‐1α inducer could alleviate the enhanced ferroptosis caused by knocking down RRM2. In vivo experiments also confirmed that overexpression of RRM2 promoted tumour growth, upregulated Ki‐67, GPX4, HIF‐1α, and N‐cadherin, and downregulated E‐cadherin. Conclusions RRM2 inhibited ferroptosis by regulating the HIF‐1α signalling pathway, thereby promoting the progression of ICC. The RRM2/HIF‐1α/ferroptosis axis may become a potential target for ICC therapy.