Xenograft tumour assay, in vivo metastasis assay and immunohistochemistry staining

Nude mice were injected subcutaneously with SW1990 cells to form the xenograft tumours. After the tumour volume reached 50 mm³, mice were divided into three groups. The tumour volume calculation formula was as follows: length × width² × 0.5. ZFAS1 siRNA, ZFAS1 siRNA plus HMGA2 plasmid or NC was injected into the interior of the tumours every 5 days for 35 days, and the tumour volume was calculated each time. The tumour tissue was dissected and photographed after 35 days. SW1990 stably expressing sh-ZFAS1 or sh-ZFAS1 plus HMGA2 plasmid were injected into the tail vein of mice. Sterile D-Luciferin firefly potassium salt (Beyotime, China) were injected into nude mice. In vivo imaging was performed by using PerkinElmer IVIS Spectrum (Xenogen, CA) and quantified by using Living Image software (Xenogen, CA). After 21 days, the lung was dissected. The subcutaneous tumour tissue and metastatic pulmonary nodules were sectioned, and immunohistochemical staining was carried out using an HMGA2 antibody (Abcam, UK). The experimental steps were performed according to the methods described previously [35]. This research was endorsed by the Research Ethics Committee of the Affiliated Hospital of Jiangnan University.