Cell transfection

HepG2 cells were cultured in 6-well plates at a density of 1×10⁴ cells/mL. After incubation for 24 hours, HepG2 cell lines were subjected to transfection with 50 nM shRNA-NC, Sh-HULC, mimic-NC, miR-377-5p mimic, or miR-377-5p inhibitor using Lipofectamine^® 3000 (Invitrogen) in line with the manufacturer’s protocol. To target HULC, pGPU6/Neo plasmid (GenePharma, Shanghai, China) was cloned from short-hairpin RNA (sh-RNA) oligonucleotides and the corresponding negative controls (14). ShRNA-NC, Sh-HULC, mimic-NC, miR-377-5p mimic, and miR-377-5p inhibitors were obtained from Shanghai GenePharma Inc. (Shanghai, China). The scrambled control shRNA (shRNA-NC) and miRNA control (mimic-NC) were used as controls. pcDNA™3.1 was bought from Invitrogen (USA). The HIF-1α pcDNA vector was constructed to overexpress HIF-1α and transfected into HepG2 cells. The cells were randomly divided into three groups, the control group, the pcDNA group, and the pcDNA-HIF-1α group. RT-PCR and Western blot were used to determine whether the overexpression was successful.