Shduxap8

shDUXAP8, shNC, human miR-485-5p mimic, and mimic NC were purchased from GenePharma (Shanghai, P.R. China). shDUXAP8 or shNC sequences were cloned into lentiviral vector pLKO.1-puro (Sigma, St. Louis, MO, USA). To produce lentivirus, lentiviral vector was co-transfected into HEK293T cells with packaging vectors using the Lentiviral Packaging Kit (OriGene, Rockville, MD, USA) according to the manufacturer’s protocol. After 48 h, lentivirus-containing supernatant was harvested from the culture, centrifuged at 500 × g for 5 min to remove cell debris, and applied to target cells in the presence of polybrene (8 μg/mL; Sigma) overnight. Then, target cells were cultured in fresh complete growth medium for 48 h and selected in medium containing puromycin (5 μg/mL) for a further 10 days.
To overexpress FOXM1 or FUS, the human FOXM1 or FUS cDNA sequence was cloned into pcDNA3.1 vector (Thermo Fisher Scientific, Waltman, MA, USA). The empty vector was used as the NC. The transfection of pcDNA3.1 vector or miRNA mimics was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.

The 4–6 weeks old BALB/c nude mice bought from Beijing Laboratory Animal Center (Beijing, China) were used in this study. A549 cells were stably transfected with short hairpin RNA against DUXAP8 (sh-DUXAP8) or sh-NC (Genepharma, Shanghai, China). Approximately 1 × 10⁶ sh-DUXAP8/sh-NC-infected A549 cells were subcutaneously inoculated into the flanks of the nude mice (5 per group). Tumor volume was detected every 3 days from 6 day using a caliper. After 30 days, the mice were euthanized and tumor weight was assessed. All experiments were performed in line with the Guide for the Care and Use of Laboratory Animals and approved by the Huaihe Hospital of Henan University Experimental Animal Ethics Committee.