Subcutaneous tumor xenograft model

Female nude mice (6–8-weeks-old) were purchased from Changzhou Kaiwen Laboratory Animal Co., Ltd. The mice were maintained under specific pathogen-free conditions and housed in ventilated cages with free access to food and water. The mice were allowed to acclimatize for one week prior to the start of the experiment. The animal procedures were performed in accordance with the guidelines of the Nanjing Medical University Laboratory Animal Center.

PANC-1 cells were cultured in 10% FBS-containing DMEM and then harvested by centrifugation with the speed of 1,000 × g for 5 min at room temperature. A total of ~4×10⁷ cells were resuspended in 100 ml saline and inoculated subcutaneously on the right flank of mice. At 40 days following inoculation, the negative control agomir (NCA), miR-23b-3p agomir (23b-3pA), or miR-23b-3p antagomir (23b-3pAnt) were injected every 3 days into the tumor, and the tumor width and length were measured every 5 days, the volume was calculated using the formula: Volume = width² × length × 0.5 (27). The mice were anesthetized with 2% isoflurane prior to euthanasia using cervical dislocation to confirm the death, and the tumors were harvested for measurement of their diameter and for histopathological investigation, 10 days later. Mice injected with 0.9% saline solution only into the right flank were used as the normal group. A total of 3 mice were used in each experimental group and the experiment was repeated three times.