Cell lines, clinical samples, animals, and reagents
The GES-1 non-malignant gastric epithelial cell line, AGS, HGC27, SGC7901 and MKN45 gastric cancer cell lines with different differentiation degrees were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and were cultured at 37 °C in 5% CO2. AGS: human gastric adenocarcinoma cell; HGC 27, human gastric cancer cell with metastatic lymph node; SGC7901: human gastric cancer cell (lymph node metastasis of patients with gastric adenocarcinoma); MKN45: human gastric cancer cell. The GES-1 and SGC7901 cells were cultured in Roswell Park Memorial Institute-1640 (RPMI)-1640 (Gibco, Rockville, MD, USA), while AGS, HGC27, and MKN45 cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco). All medium were supplemented with 10% fetal bovine serum (Hyclone, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (100 mg/mL) (Invitrogen, Carlsbad, CA, USA).
35 paired GC tissues and adjacent normal tissues from GC patients who underwent surgical resection were included in this study. In the 35 GC cases, there were 20 males and 15 females with a median age of 64.2 years (range 23–87 years). This study was approved by the ethics committee of the Lianyungang Municipal Oriental Hospital (Liangyungang, Jiangsu, China). In addition, we obtained informed consents from each patient for experiment.
6–8 weeks female BALB/C nude mice were purchased from the experimental animal center of Shanghai, China and fed in a pathogen-free mice facility, which were cultured at 22–25 °C temperature and 40–60% humidity, respectively. All animal study protocol was approved by our hospital ethical committees.
Cisplatin and Erk inhibitor Ly3214996 were purchased from Sigma (Sigma, USA, Cat#:BP809 for cisplatin and A668057 for Ly3214996) and stored at 10 mmol/L stock in DMSO solution.
Cell transfection and infection
Expression plasmids encoding BANCR and corresponding empty vectors as control were obtained from GenePharma Co. Ltd. (Shanghai, China). Three BANCR siRNAs were also obtained from GenePharma Co. Ltd. We transfected BANCR plasmids or BANCR siRNAs into GC cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.
RNA isolation and qRT-PCR
Total RNA was isolated from GC tissue samples or cell lines by TRIzol® (Invitrogen) following the manufacturer’s protocol. The complementary DNAs (cDNA) were synthesized using First Stand cDNA Synthesis kit (Roche, Basel, Switzerland), and then performed quantitative PCR analysis by Applied Biosystems™ PowerUp™ SYBR™ Green Master Mix kit (Thermo Fisher Scientific, Waltham, MA, USA in Applied Biosystems QuantStudio™ 12 K Flex Software (Thermo Fisher Scientific). The primer sequences were as follows: BANCR forward, 5’-ACAGGACTCCATGGCAAACG-3’ and BANCR reverse, 5’-ATGAAGAAAGCCTGGTG-3’; GAPDH forward, 5’-ACCACAGTCCATGCCATCAC-3’ and 5’-TCCACCACCCTGTTGCTGTA-3’. GAPDH acted as internal standard. The BANCR relative quantitative expression was calculated by the 2−ΔΔCt methods.
Cell viability assays
We performed cell viability analysis with a cell Counting Kit-8 (CCK-8, DoJinDo, Tokyo, Japan) according to the manufacturer’s protocol. Briefly, 5000 cells/well were seeded in 96-well plates and treated with cisplatin at different concentration gradient for 24 hours. Finally, we measured the absorbance at 450 nm to determine cell viability.
Colony formation assays
We seeded GC cells (500/well) into 6-well plates and treated with cisplatin for 12 days. We added fresh medium into the plates every 3 days. After 12 days, we fixed colonies with 4% paraformaldehyde and then stained the colonies with 1% crystal violet. Finally, we photographed and counted the cell colonies.
The total protein from cultured cells and tumors were obtained with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and separated by 10% polyacrylamide gel electrophoresis, followed by transferring onto polyvinylidene fluoride membranes (Millipore, MA, USA). The protein membrane was incubated with primary antibodies (Anti-pERK1/2, anti-total ERK1/2, Anti-pMEK1/2, Anti-MEK1/2, and Anti-GAPDH antibody, Abcam, Cambridge, MA, USA) overnight at 4 °C. We used the enhanced chemiluminescence (Thermo Scientific, Waltham, MA) to detect protein bands and semi-quantified the band density with an ImageJ analysis software (National Institutes of Health).
Xenograft mouse model
The lentivirus particles carrying BANCR siRNA-1 sequences (si BANCR-1) were purchased from GenePharma Co. Ltd. We obtained the MKN45 cells with BANCR stably knock-down by infection with si BANCR-1 lentivirus. Then we subcutaneously injected 5 × 106 MKN45 cells with BANCR stably knock-down or control cells into the back flank of female nude mice (6–8 weeks old). Cisplatin (40 µM/kg) was administered intraperitoneally at day 5, 8, 12 and 15. Tumor size was measured at the indicated days by using a calliper. After 19 days, we isolated the xenograft tumor from mice, photographed the tumor and measured its weight.
All data were presented as the mean ± standard deviation (SD). We performed statistical analysis by one tail Student’s t test. A p-value less than 0.05, 0.01 or 0.001 were significantly considered difference.