Six-week-old athymic nude mice were obtained from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). Mice were divided into five groups: sham group, metformin group, metformin + NC group, metformin + miR-324-3p overexpression group, and metformin + miR-324-3p knockdown group (n = 6 in each group). Mice were injected with 3 x 10⁶ MDA-MB-231 cells subcutaneously into the right flank. For the miR-324-3p overexpression or knockdown in the mice, two groups of mice were treated with miR-324-3p overexpression or knockdown lentivirus (GenePharma), respectively, by intratumoral injection of 50 ul of lentivirus (4 x 10⁷ IU/ml) after the tumor cell injection. One day after tumor cell inoculation, the sham-treated group was treated with PBS and metformin-treated groups were treated with 200 mg/kg metformin every 2 days through intraperitoneal injection.

A total of 30 male BALB/c nude mice (4-6 weeks old; 18-23 g) were randomized into four groups (7-8 mice per group): i) control group; ii) treated with 15 mg/kg ICS II; iii) treated with 25 mg/kg ICS II; and, iv) treated with 35 mg/kg ICS II. ACHN and Caki-1 cells (1 x 10⁷) were suspended in 50 ul MEM media mixed with 50 ul Matrigel (BD Biosciences) and injected subcutaneously into the right flank of mice with 1.5%pentobarbital sodium (60 mg/kg body weight; intraperitoneal injection) under anesthesia. Weight lossof more than 20% was considered a humane endpoint.