Subcutaneous xenograft model and lung metastasis model were established to assess the growth and metastasis ability of cells in vivo. Male BALB/C nude mice (4-week-old) were purchased from Shanghai SLAC Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China) and randomly divided into four groups (n = 6 per group). HCCLM3 cells transfected with sh-MAPKAPK5-AS1 and sh-control and Hep3B transfected with MAPKAPK5-AS1 and Vector were applied in our animal experiments. For subcutaneous xenograft model, transfected HCCLM3 and Hep3B cells were subcutaneously injected into the left flank of the mice, respectively (1 × 106 per injection) (six mice per group). Tumor size was measured every 7 days, and tumor volume was calculated by the formula: tumor volume = 0.5 × (long diameter) × (short diameter)2. Tumors were removed for measurement after 4 weeks. For lung metastasis model, transfected HCCLM3 and Hep3B cells were injected via the tail vein (1 × 106 per injection) (five mice per group). After 6 weeks, mice were sacrificed and the lung metastases were confirmed by hematoxylin and eosin (H&E) staining. The animal experiments were approved by the Animal Care and Use Committee of Xi’an Jiaotong University.

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