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Metformin is a widely prescribed hypoglycemic drug.Many studies have shown its anti-cancer properties.In the present study,we aimed to explore the effect of metformin on breast cancer and clarify the underlying mechanism.Our results showed that metformin induced ferroptosis in MDA-MB-231 cells through upregulating miR-324-3p expression.Overexpression of miR-324-3p inhibited cancer cell viability.miR-324-3p inhibitor promoted cell viability.Further studies showed that the effect of miR-324-3p was mediated by directly targeting glutathione peroxidase 4(GPX4).miR-324-3p bound to the 3'-UTR of GPX4 and led to the downregulation of GPX4.In vivo studies showed that metformin induced ferroptosis by upregulating miR-324-3p in the xenograft model of breast cancer in mice.Our study suggested that metformin promotes ferroptosis of breast can-cer by targeting the miR-324-3p/GPX4 axis.Metformin could act as a potential anti-cancer agent through the induction of ferroptosis.