Fig. 2
PSMA3-AS1 knockdown restrains cell proliferation, migration, and invasion of GC cells. (A) A pcDNA-3.1 vector containing full-length PSMA3-AS1 and a pRNA-H1.1 vectors carrying sh-PSMA3-AS1 were constructed via the Hind III and BamH I sites. PSMA3-AS1 overexpression plasmid was transfected into HGC-27 cells, while PSMA3-AS1 knockdown plasmid was transfected into AGS cells using Lipofectamine 2000. The stable transfectants were selected by G418 (400 µg/ml) for 6 weeks. PSMA3-AS1 levels in stable transfectants were measured by real-time PCR (n = 3). GAPDH served as an internal control. (B) The copy numbers of PSMA3-AS1 in stable transfectants were determined by ddPCR. Results are expressed as copies/µl (n = 3). (C) The stable transfectants were seeded on 96-well plates at 3000 cells/well and cultured for 0 − 72 h. The optical density at 450 nm was monitored to evaluate cell proliferation by a CCK-8 assay (n = 3). (D) The stable transfectants seeded on 6-well plates (500 cells/well) and allowed to grow for 2 weeks at 37 °C to form visible colonies. After staining, colony numbers were counted and colony formation rate was calculated using the formula described in Materials and Methods section (n = 3). (E) The stable transfectants were harvested and subjected to flow cytometric analysis of cell cycle distribution (n = 3). (F) Total proteins were extracted from stable transfectants. The levels of cyclin D1 and CDK were determined by western blotting (n = 3). GAPDH served as an internal control. (G) The stable transfectants (5 × 105 cells) were seeded on 6-well plates. After adhering to the plates, the cell monolayer was scratched and further cultured for 24. Wound healing rate (%) was calculated using the formula described in Materials and Methods section (n = 5). (H) Cell invasion was evaluated by transwell assay. The numbers of invaded cells were counted in five random fields, and the average numbers of invaded cells were calculated (n = 5). *P < 0.05 and **P < 0.01 compared with sh-NC or vector. Student′s t-test was used to compare two groups