Establishment of subcutaneously xenografted tumor mice model

Twelve nude mice (species: BALB/c; sex: male; age: 6 weeks; weight: 20 ± 2 g) were bought from SLAC (Shanghai, China). All the mice were fed in SPF grade feeding rooms under a 12 h light/dark cycle with free access to food and water. After 4 days of feeding, the mice were randomly divided into four groups: the shNC group (n = 3), the shPCAT6 group (n = 3), the MC group (n = 3), and the mimic group (n = 3).

In short, 2 × 10⁶ GH3 cells which were stably transfected with shNC, shPCAT6, MC, or miR-139-3p mimic were resuspended in 100 μl of PBS. Then the cell-containing PBS was mixed with 100 μl of Matrigel (354248, Corning). Subsequently, the cells in the mixed solution were subcutaneously injected into the right forelimb (above axillary fossa) of the nude mice in the four groups. Then the mice were normally fed and the tumor of the mice was observed every day. The tumor volume was measured and calculated by the formula: tumor volume (mm³) = major diameter (L) × (minor diameter)² (W²)/2. Thirty-five days later, the mice were anesthetized with 2% sodium pentobarbital (B005, Jiancheng) at the dose of 50 mg/kg and then sacrificed by cervical dislocation. Finally, the tumor tissues were photographed, weighed, and stored for later use.