In vivo xenograft mouse model

Female BALB/c nude mice at 4–6 weeks of age and weighing 18–20 g were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. The Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine approved the animal experiments. HT29 cells in the logarithmic growth phase were collected, adjusted to a density of 2.5 × 10⁷ cells/ml, and injected into the subcutaneous tissue of nude mice at a volume of 0.1 ml per mouse. Overall, 15 mice were randomly divided into three groups with 5 mice in per group (the control group, the tumorigenic group without 5-FU treatment, and the tumorigenic group with 5-FU treatment) and raised in a specific-pathogen-free (SPF) environment. Animals in the two experimental groups were inoculated with tumor cells. After 1 week, when the xenografted tumors had grown to a certain size (approximately 100 mm³), animals in one group were intraperitoneally injected with 5-FU (20 mg·kg^− 1·d^− 1) every 3 days, while animals in the other group were treated with the same amount of saline. The length and width of the tumors were measured every 3 days by a researcher who was unaware of the group allocation until the experiment was ended. The tumor volume (V) was calculated using the following formula: V = length/2 × width². The mice were placed in a 10 L chamber with 99% CO₂ (3 L/min) for 5–10 min, and vital signs (including breath, heartbeat, and muscular tension) were closely monitored until death was confirmed. Serum samples were collected, and circulating miR-1290 levels were verified by RT-qPCR.