Mgc 803

Human gastric carcinoma cell lines (MGC-803, AGS, and BGC-823), originally purchased from Cell Resource Center, Chinese Academy of Medical Sciences (CAMS, Beijing, China), were cultured in RPMI 1640 (Gibco, Grand Island, USA) (for MGC-803 and AGS cells) or F12 medium (Gibco, Grand Island, USA) (for BGC-823 cells) supplemented with 10% fetal bovine serum, in a humidified, 5% CO₂ incubator at 37°C. Next, 1×10⁴ cells were plated in 48-multiwell plates (Corning, NY, USA) and grown to 80–90% confluence for transient transfection using Lipofectamine 2000 Reagent (Life Technologies, Inc., Rockville, USA) according to the manufacturer’s protocol. Cells were then co-transfected with 500 ng of pA₋₆₃₈-C₋₃₅₈, pA₋₆₃₈-T₋₃₅₈, pG₋₆₃₈-C₋₃₅₈, and pG₋₆₃₈-T₋₃₅₈, or PGL3-basic plasmids and 1.0 ng of Renilla luciferase reporter plasmid pRL-SV40 (Luciferase Assay System, Promega, Madison, USA) for standardization of the transfection efficiencies. Luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega, Madison, USA) on a TD-20/20ⁿ Luminometer (Turner Designs, Promega, Madison, USA) according to the manufacturer’s protocol. Results were normalized for Renilla activity and were expressed as relative luciferase activity (RLA). Three independent transfection experiments were performed, and each was done in triplicate.

Paired samples were obtained from patients undergoing surgery for gastric cancer from The General Hospital of the People’s Liberation Army and Cancer Institute and Hospital, Chinese Academy of Medical Sciences. The samples were immediately frozen and stored in liquid nitrogen until RNA extraction. The use of the tissue samples for all experiments was approved by the ethical board of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (IBMS, CAMS). Specifically, all participants provided their verbal informed consent to participate in this study and were compensated for some money. Their verbal informed consents were written down. This consent process was discussed and approved by the ethics board. We collected the clinical samples in accordance with the approved guidelines.
The MGC-803, HGC-27, MKN-45, and SGC-7901 cell lines were purchased from the Cell Resource Center of IBMS, CAMS. MGC-803 was propagated in DMEM (Life Technologies, CA, USA), supplemented with 10% fetal bovine serum (FBS; PAA, Pasching, Austria) and streptomycin (100 μg/ml), penicillin (100 U/ml). The HGC-27, MKN-45, SGC-7901 were maintained in RPMI 1640 medium (PAA) supplemented with 10% FBS (PAA).

The human GC cell lines (MGC-803, SGC-7901, MKN-45 and HGC-27) and one normal gastric mucosa cell line (GES) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Four of the cell lines (SGC-7901, MKN-45, HGC-27 and GES) were cultured in Roswell Park Memorial Institute (RPMI)-1640 (PAA) containing 10% v/v fetal bovine serum (FBS; Trace Scientific, Melbourne, Australia), the other cell line MGC-803 in Dulbecco's modified Eagle medium (Gibco; Invitrogen; Life Technologies, Germany) containing 10% FBS. All the cell lines were incubated at 37°C in a humidified atmosphere with 5% carbon dioxide.

Four human gastric cancer cell lines (BGC-823, SGC-7910, MGC-803 and AGS) and a normal human gastric mucosa cell line (GES 1) were received from BeNa Culture Collection (Beijing, China). Cells were kept at 37°C in a humid atmosphere with 5% CO₂. BGC-823 cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Invitrogen, CA, USA). SGC-7910 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI1640) medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% FBS (Invitrogen, CA, USA). MGC-803 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM – 214.5 g/L glucose) containing 10% FBS (Invitrogen, Carlsbad, CA, USA). AGS cells were cultured in Nutrient Mixture F-12 (F-12) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (Invitrogen). GES 1 cells were cultured in RPMI medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Invitrogen).