Si fen1

The human MG-63, U2OS and 143B cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, USA.) and 1% penicillin and streptomycin and maintained at 37°C in a humidified incubator with 5% CO₂. The miR-193b mimic, miR-193b inhibitor, and negative control were designed and synthesized by Guangzhou RiboBio Co., Ltd (Guangzhou, China). The small interfering RNA (siRNA) targeting FEN1 (si-FEN1) and negative control (NC) siRNA were also synthesized by Guangzhou RiboBio Co., Ltd. Plasmids for FEN1 and NC overexpression were purchased from Shanghai GenePharma Co., Ltd. All plasmid constructs were verified by sequencing. Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for miRNA or siRNA transfection into OS cells according to the manufacturer’s protocols. Cells were harvested for subsequent experiments 48 h after transfection.

American Type Culture Collection (ATCC, Manassas, VA, USA) provided the human MG-63, U2OS and 143B OS cell lines. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Carlsbad, CA, USA) and maintained at 37°C in a humidified incubator with 5% CO^{2 (link)}. The miR-610 mimic, miR-610 inhibitor, and negative control were designed and synthesised by Guangzhou RiboBio Co., Ltd (Science City, Guangzhou, China), along with the small interfering RNA (siRNA) targeting FEN1 and IGF-1R (si-FEN1, si-IGF1R) and negative control (NC) siRNA. All plasmid constructs were verified by sequencing. Lipofectamine 2000 (Invitrogen, USA) was used for each miRNA or siRNA transfection according to the manufacturer’s protocol. Cells were harvested for subsequent experiments 48 h after transfection or anti-cancer drug treatment [Metformin 10 mM, ADM (0.7095 μM for MG-63, 0.9787 μM for U2OS), DDP (0.9731 μM for MG-63, 1.018 μM for U2OS), MTX (35.68 μM for MG-63, 33.54 μM for U2OS)]. Metformin, ADM, DDP and MTX were purchased from Sigma-Aldrich (St. Louis, MO, USA). The drugs were prepared immediately before use. The FEN1, IGF-1R, cleaved caspase-3, and GAPDH antibodies were obtained from Abcam (Cambridge, MA, USA).