circFADS2 inhibits ferroptosis associated with IGF2BP2-dependent SLC7A11 m6A modification in colorectal cancer cells.

Abstract

Background: Colorectal cancer (CRC) is one of the most common malignancies worldwide, and its pathogenesis is highly complex. The aim of this study was to explore the mechanism of action of circFADS2- and IGF2BP2-mediated SLC7A11 m6A modification in CRC.

Methods: In vitro experiments were conducted to knock down circFADS2 and overexpress SLC7A11 in CRC cells, and circFADS2 expression was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR); Cell Counting Kit-8 (CCK-8) and colony formation experiments were used to detect cell proliferation; cell migration was detected by Transwell; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used for detection of cell apoptosis; western blot (WB) was employed for detection of SLC7A11 expression; the levels of malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), and Fe²⁺ levels were detected; and messenger RNA (mRNA) stability testing, nuclear cytoplasmic separation experiments, RNA immunoprecipitation, fluorescence in situ hybridization (FISH), and immunofluorescence (IF) were used to verify the binding and mechanism of action of circFADS2, IGF2BP2, and SLC7A11 mRNA. In vivo experiments were conducted by injecting CRC cells from each group subcutaneously into the right side of mice, and the growth of tumor cells was measured in each group in vivo.

Results: After knocking down circFADS2, the expression of circFADS2 was downregulated in CRC cells. There was a significant reduction in cell proliferation and migration and a significant increase in cell apoptosis. The expression of SLC7A11 was significantly reduced; MDA content significantly decreased; GSH levels decreased; ROS levels increased; and the concentration of Fe²⁺ significantly increased. After circFADS2 knockdown, the growth of CRC cells in vivo was inhibited. In addition, mRNA stability testing showed that circFADS2 knockdown significantly reduced the stability of SLC7A11 mRNA. The nuclear cytoplasmic separation experiment showed that circFADS2 was mainly expressed in the cytoplasm. RNA immunoprecipitation indicated a binding relationship between IGF2BP2 and circFADS2, as well as between IGF2BP2 and SLC7A11 mRNA. The results of FISH and IF analysis showed that circFADS2, IGF2BP2, and SLC7A11 mRNA were co-localized with IGF2BP2 in the cytoplasm.

Conclusions: circFADS2 facilitates the formation of the circFADS2/IGF2BP2/SLC7A11 mRNA-protein complex, thereby enhancing the m6A methylation of SLC7A11. This process significantly promotes ferroptosis in CRC cells.