Cell culture and transfection

A total of four NPC cell lines 5-8F, CNE2, CNE1, and HONE-1 and one immortalized human nasopharyngeal epithelial cell line NP69 (American Type Culture Collection [ATCC), Manassas, VA, U.S.A.) were incubated in an incubator containing RPMI-1640 complete medium consisting of 10% fetal bovine serum (FBS), 100 μg/ml streptomycin and 100 U/ml penicillin at 37°C with 5% CO₂ and 95% saturated humidity with the medium replaced 3–4 times per week depending on the cell growth. Cells were sub-cultured when the cell confluence reached about 80%. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was carried out to measure the level of miR-613 in each cell line in order to screen out two cell lines with the lowest miR-613 level for following cell experimentations.

CNE1 and HONE-1 cells were classified into blank (cells without any transfection), negative control (NC)-mimic (cells transfected with miR-613 NC sequence), miR-613 mimic (cells transfected with miR-613 mimic), si-NC (cells transfected with si-NC), si-FN1 (cells transfected with si-FN1), miR-613 mimic + overexpression (oe)-FN1 (cells transfected with miR-613 mimic and oe-FN1) and LY294002 groups (cells treated with 40 μmol/L LY294002, the inhibitor of the AKT signaling pathway). The target plasmids were purchased from Dharmacon (Lafayette, CO, U.S.A.). CNE1 and HONE-1 cells in logarithmic growth phase were inoculated into a 6-well plate at a density rate of 3 × 10⁵ cells/ml. When cell confluence reached 80%, cells were transfected using lipofectamine 2000 kits (Invitrogen, Carlsbad, California, U.S.A.). A total of 4 μg the target plasmid and 10 μl lipofectamine 2000 were respectively diluted using 250 μl serum-free Opti-MEM (Gibco, Carlsbad, California, U.S.A.), mixed gently, and allowed to stand for 5 min at room temperature. After that, above two mixtures were evenly mixed and allowed to stand for 20 min. The mixture was then added to the culture wells and cultured in an incubator with 5% CO₂ at 37°C. After 4 h, with medium changed to complete medium, cells continued to be cultured for 48 h and were collected for subsequent experiments.