HepG2 cell transfection

HepG2 cells were cultured in RPMI 1640 medium containing 10% FBS at 37° C with 50 mL/L CO₂. When cells reached 90% confluence and were in the logarithmic growth phase, cells were trypsinized to prepare cell suspension (2.5 × 10⁴ cells/mL), which was seeded into a 6-well plate at 2 mL/well. When cells reached 30% confluence and were in the logarithmic growth phase, different lentivirus was added (2 × 10⁶ TU lentiviruses were mixed with 5 μg of polybrene and 1 mL serum-free and antibacterial-free medium). Transfection was observed under an inverted fluorescence microscope after two or three days. After transfection for two days, 1 μg/mL of puromycin was added to each well to screen stably transfected cells. Cells were then allowed to grow in culture to obtain stable transfected cells. After successful lentivirus infection, cells at 70-80% of confluence were selected and subjected to transient transfection of miR-222-5p mimic or inhibitor using the liposome Lipofectamine^TM 2000 kit as per instructions (Invitrogen, Carlsbad, CA, USA). After two days, cells were harvested for subsequent experiments.

Plasmids, and lentivirus clone vectors pLKO.1-TRC (plasmid #10878) and pcDNA3.3-SOX2 (plasmid, #26817) were provided by Addgene (Cambridge, MA, USA). Double-stranded oligonucleotide short hairpin RNA (shRNA) was cloned at the AgeI/EcoRI site in the pLKO.1-TRC lentiviral vector [22]. HepG2 cells were treated with overexpression empty lentivirus vector [overexpression-negative control (oe-NC)], sh-NC lentivirus, sh-SOX2 lentivirus, sh-CCAT1 lentivirus, oe-CCAT1 lentivirus, oe-SOX2 lentivirus, oe-EGFR-1 lentivirus at doses of 0.5, 1.0, and 1.5 μg, 1.5 μg oe-EGFR-3 lentivirus, DMSO, 1 μM gefitinib (EGFR-inhibitor), mimic-NC, miR-222-5p mimic, inhibitor-NC, miR-222-5p inhibitor, sh-EGFR lentivirus, small interfering (si)-NC lentivirus, and si-CYLD lentivirus, alone or in combination. After 48 hours of transient transfection, expression levels in HepG2 cells were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) to evaluate transfection efficiency.