miR-28-5p inhibits carcinogenesis in colon cancer cells and is necessary for erastin-induced ferroptosis

BACKGROUND: Ferroptosis is a newly discovered type of regulated cell death, the underlying mechanisms of which need to be further illuminated. The regulatory activity of miR-28-5p in ferroptosis in colon cancer cells is currently unclear. This study set out to investigate the effect of miR-28-5p on ferroptosis in colon cancer cells and determine its underlying mechanism. METHODS: Biochemical Kits were used to measure iron concentration, malondialdehyde (MDA) concentration, glutathione (GSH) concentration and glutathione peroxidase (GPX) vitality. Cell counting kit 8 (CCK8) assays were conducted to evaluate cell viability. Flow cytometry was conducted to assess apoptosis. Transwell™ assays were used to measure the migratory and invasive abilities of HCT116 cells. Western blotting was used to measure the protein relative expression of NEDD4 binding protein 1 (N4BP1). Quantitative real-time polymerase chain reaction (RT-PCR) was used to measure the RNA relative expression of N4BP1 and miR-28-5p. RESULTS: Ferroptosis was induced in HCT116 cells by erastin in a dose- and time-dependent manner, which caused significant inhibition of proliferation, migration, and invasion in HCT116 cells; however, there was no obvious effect on apoptosis. miR-28-5p expression was decreased in colon cancer cells compared with the normal colon cells but was upregulated in erastin-treated HTC116 cells. Additionally, when overexpressed via the transfection of miR-28-5p mimics, miR-28-5p had an inhibitive effect on proliferation, migration, and invasion, while promoting apoptosis, in HCT116 cells. erastin-induced ferroptosis was also increased by miR-28-5p overexpression. Compared with normal colon cells, following erastin treatment, NEDD4 binding protein 1 (N4BP1) expression was increased in colon cancer cells and further decreased in HTC116 cells. miR-28-5p overexpression also inhibited N4BP1 mRNA and protein expression in HTC116 cells. CONCLUSIONS: miR-28-5p plays an important role in ferroptosis by targeting N4BP1 and could serve as a potential therapeutic approach for colon cancer.