Sh circ 1

Normal osteoblast hFOB1.19 and OS cell lines (HOS, U2OS, and SAOS-2) were purchased from the American Type Culture Collection (Manassas, VA, USA). All cells were kept in an incubator with 5% CO₂ at 37 °C and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Grand Island, NY, USA) complemented with fetal bovine serum (FBS, 10%, Sigma, Louis, Missouri, MO, USA), streptomycin (100 μg/mL, Sigma), and penicillin (100 U/mL, Sigma).
Short hairpin RNA targeting circ_0005909 (sh-circ#1 and sh-circ#2) and negative control (sh-NC) were bought from RiboBio (Guangzhou, China). MiRNA mimics and inhibitors targeting miR-936 (miR-936 and anti-miR-936) and their negative controls (miR-NC and anti-NC) were procured from RiboBio. The sequence of HMGB1 was cloned into the pcDNA3.1 vector (vector) (Invitrogen, Carlsbad, CA, USA) to construct the overexpression vectors for HMGB1. When the cells were cultured to approximately 80% confluence, OS cells (HOS and U2OS) were transiently transfected with the assigned oligonucleotides or vectors using Lipofectamine 3000 reagent (Invitrogen) and cultured for 48 h.

The animal experiment was ratified by the Ethics Committee of Henan Provincial People’s Hospital. 8 BALB/c nude mice (athymic, 4–5 weeks old) were purchased from Charles River Laboratories (Beijing, China) and were fed under specific-pathogen-free conductions. HOS cells (1 × 10⁶) with sh-NC or stable lentivirus-mediated sh-circ_0005909 (sh-circ#1, RiboBio) were subcutaneously injected into the dorsal side of the nude mice (4 mice/group). The tumor volume was measured once a week using the equation: Volume = (length × width²)/2. The mice were euthanized on day 35 after injection for subsequent analysis.