Ten male BALB/c nude mice (6 weeks old, 20–25 g) were bought from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were divided into two groups (5 mice/group) and hypodermically injected with 3 × 106 stable HCT116 cells (sh-NC group, sh-circ group). Tumor length (L, mm) and weight (W, mm) were weekly measured by a vernier caliper and tumor volume (mm3) was expressed as: L (mm) × W2 (mm2)/2. After 4 weeks, mice were sacrificed by gradually increasing the concentration of CO2 (flow rate, 30% of volume/min) in the sealed container. Tumors were then excised and weighed, followed by the RNA and protein extraction for qRT-PCR and Western blot detection. All animal operations were in accordance with the Animals Guidelines of National Institutes of Health and this assay was ratified by Animal Ethics Committee of Jingmen No.1 People’s Hospital.

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