The Xenograft Experiment in Nude Mice

Sixty nude mice (6 weeks-old, body weight: 20-22g) were selected as the experimental subjects to establish the xenograft model. All mice were housed and maintained under specific pathogen-free conditions free of water and food. CRC cells (SW480 and HCT116) stably transfected with MIR31HG overexpression plasmids and their negative controls were selected, and the blank control group was set. The cells were adjusted to reach a density of 2×10⁸ cells/mL when they were in the logarithmic growth phase. Afterward, 0.1 mL cell suspension was subcutaneously injected into the left forelimb armpit of each nude mouse. Within 4 weeks after the injection, the survival status and body weight of the mice, as well as the number of lung metastases, were monitored. Meanwhile, the weight, the longest diameter (a) and shortest diameter (b) of the tumor stripped from the newly dead mice were measured. Tumor volume (V)=0.5×a×b². For evaluating the lung metastasis of CRC cells, CRC cells (SW480 and HCT116) stably transfected with MIR31HG overexpression plasmids and their negative controls were injected into the tail vein of the nude mice. Four weeks later, the mice were euthanized and their lungs were removed. The images and number of metastatic nodules in the lungs were captured and counted. Moreover, immunohistochemistry was conducted to detect VEGFA (1:200, ab52917) and KI67 (1:150, ab15580) in the formed tumor tissues according to previous study (24). The antibodies were all bought from Abcam company. The primary antibodies include All experiments were approved by the Animal Care and Use Committee of Fouth Affilated Hospital of Anhui Medical University and performed in accordance with institutional guidelines.