Xenograft model

Ten healthy BALB/c nude mice (5-week-old, female, 16–20 g) from the same animal cage were purchased from Vital River Laboratory (Beijing, China), and animal study obtained the approval of the Animal Ethics Committee of Changsha Central Hospital. Animals were adaptive feeding for three days under standard laboratory conditions (specific-pathogen-free, 23°C ± 2°C, 45%~65% humidity) and randomly divided into two group (n = 5/group). H1650 cells transfected with sh-NC or sh-circ-OXCT1 were subcutaneously inoculated into the dorsal side of the mice after anesthesia with 2% isoflurane. Tumor volume was monitored via the method of length×width² × 0.5 every 7 d for 35 d. On the 35th day after cells injection, after mice were euthanized by overdose CO₂ for 10 min, tumors were taken out and weighed. To measured RNA and protein expression in tumor tissues of nude mice, qRT-PCR and western blotting were performed. In addition, immunohistochemistry (IHC) assay was conducted to detect the expression of SLC1A5 and ki-67. Antibodies used in IHC were listed in Table 2. In this work, the potential confounders were not controlled. Dead and ailing animals were excluded from the study. There were no experimental animals were excluded in this study because there were no dead or ailing animals. Each experimental group, report any animals, experimental units or data analysis were no exclusions. The data distribution of all animal experimental results was normal, and comparison was analyzed by Student’s t-test.