Sh con

Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used according to the manufacturer's protocol. The miR-365a-3p mimic, control inhibitor, miR-365a-3p inhibitor, inhibitor NC, pcDNA-PGK1 and pcDNA-NC were all obtained from Gene-Pharma (Shanghai, China). Lentiviral infection was employed to generate stably transfected OS cell lines. The plasmid for HCG18 knockdown (sh-HCG18) and the empty plasmid (sh-CON) were obtained from Gene-Pharma (Shanghai, China). To establish stable HCG18-knockdown cell lines, the target cells were co-infected with 1 × 10⁸ lentivirus transducers and polybrene (Sigma-Aldrich, St. Louis, MO, USA). Furthermore, 2.5 μg/mL of puromycin was used to screen the infected cells after 72 h.

CircCTNNA1 small interfering RNAs (siRNAs), overexpressing vector and short hairpin RNA (shRNA) (si-circCTNNA1#1/#2, circCTNNA1 and sh-circCTNNA1), miR-363-3p mimic and inhibitor (miR-363-3p and in-miR-363-3p), pcDNA CXCL5 overexpression plasmid (CXCL5), or their negative controls (si-con, vector, sh-con, miR-con, in-miR-con and pcDNA) were obtained from Genepharma (Shanghai, China). SW620 and SW480 cells were seeded into 24-well plates, and transfection was conducted using Lipofectamine 3000 reagent (Invitrogen) when the cells reached 50–60% confluences.

The lentiviral vectors carried shRNA for circFBXL5 (sh-circFBXL5) or negative control (sh-con) were generated via GenePharma, and transfected into MDA-MB-231/5-FU cells. The non-transfected cells were regarded as mock group. Female BALB/c nude mice (5-week-old; Charles River, Beijing, China) were arbitrarily divided into mock, sh-con or sh-circ_0006916 groups (n = 6/group). Transfected or non-transfected MDA-MB-231/5-FU cells (5 × 10⁶ cells) were subcutaneously injected into the right flank of nude mice. The tumor size was examined every 7 days. The volume was calculated via length × width²/2. All mice were euthanized via 5% isoflurane after cell injection for 28 days. Tumor tissues were weighed and harvested for detection of circFBXL5, miR-216b and HMGA2 expression. This experiment was approved via the Animal Ethical Committee of the First Affiliated Hospital of Zhengzhou University and conducted under the National Institutes of Health.