SGC-7901 (Shanghai Institute of Cell Research, Shanghai, China), a moderately differentiated human gastric adenocarcinoma cell line, was incubated in RPMI 1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou, China) at 37℃ in 5% CO2 incubator.
Three shRNA-expressing lentiviral vectors (LV) including LV-negative control (LV-NC), LV-CXCR7-1 and LV-CXCR7-2 were from Cyagen Biosciences. CXCR7-siRNA-1 was 5’-CGCACTGCTACATCTTGAA-3’, CXCR7-siRNA-2 was 5’-GCCGTTCCCTTCTCCATTATC-3’. In the presence of polybrene (5 μg/ml), the SGC-7901 cells infected with these three kinds of LV were selected by puromycin (1.5 μg/ml) (Sigma-Aldrich, Saint Louis, MO, USA). Reverse transcription quantitative real-time PCR (RT-qPCR) and western blot were used to detected the mRNA and protein expression.
Reverse transcription quantitative real-time PCR (RT-qPCR)
RNA extraction kit (Fastagen, Shanghai, China) was used to extract total RNA according to the manufacturer’s instruction. The RNA purity and concentration were measured with Thermo Scientific NanoDrop spectrophotometer. cDNA was synthesized according to the instructions of PrimeScriptTM RT Master Kit (TaKaRa, Otsu, Japan). The required primer sequences are listed in Table 1. SYBR Premix Ex TaqTM II kit (TaKaRa, Otsu, Japan) was used to perform RT-qPCR according to the manufacturer’s protocol. The applied PCR conditions were: preliminary denaturation at 95℃ for 30 s, followed by 40 cycles at 95℃ for 5 s and 60 ℃ for 30 s. The 2-△△CT (CT: threshold cycle) method was used to calculate the RNA expression levels.
RIPA lysis buffer (Beyotime, China) was used to extract total protein according to the manufacturer’s instruction. The total protein sample was separated by SDS-PAGE, transferred to a PVDF membrane (Millipore, Billerica, MA, USA), blocked in non-fat milk, incubated with primary antibodies at suitable dilution concentration followed by secondary antibody. The protein bands were detected using an ECL plus chemiluminescence detection kit (Millipore, Billerica, MA, USA). Gel-pro Analyzer 4.0 software (Media Cybernetics, CA, USA) was used to analyze the integral optical density (IOD). Polyclonal antibodies against CXCR7, MMP-2, MMP-9, VEGF, E-cadherin, N-cadherin, vimentin, snail, β-actin and secondary antibody were purchased from Santa Cruz, Dallas, TX, USA. Monoclonal antibodies against p-Akt and t-Akt were purchased from Cell Signaling Technology, Danvers, MA, USA.
Cell migration assay
Serum starved LV-NC and LV-CXCR7-1 cells were resuspended at a cell density of 5×105 cells/ml in medium containing 1% FBS. 200 μl of the cell suspension was added to the upper chamber of a 24-well transwell, and 600 μl of medium containing 10% FBS with or without SDF-1 (100 ng/ml) was added into the lower chamber. There were four experimental groups: LV-NC, LV-NC+SDF-1, LV-CXCR7-1 and LV-CXCR7-1+SDF-1. After 48 h of incubation, the cells were washed with PBS, fixed with methanol for 10 min and washed with PBS again. The chamber was stained with crystal violet for 20 min and then washed with PBS. The cells on the upper transwell chamber surface that failed to perforate the chamber were gently wiped off with a cotton swab. The perforating cells on the lower chamber surface were observed and pictured under an inverted microscope (Nikon, Japan). Five fields of view were randomly selected to count the number of perforating cells; the average count was calculated.
Cell invasion assay
50 μl of diluted Matrigel (BD Biosciences, Bedford MA) was added to the upper chamber of the transwell. The chamber was cultured overnight at 37℃ in a 5% CO2 incubator to reconstitute the basement membrane. On the next day, serum-starved LV-NC and LV-CXCR7-1 cells were resuspended at cell density of 5×105 cells/ml in medium containing 1% FBS; 200 μl of the cell suspension was added to the upper chamber of a 24-well transwell coated with Matrigel, and 600 μl of medium containing 10% FBS with or without SDF-1 (100 ng/ml) was added to the lower chamber. The culture plates were transferred to an incubator for 48 h. Thereafter, the procedures were the same as those described for the cell migration assay. The number of perforating cells among the four groups was compared.
SPSS version 13.0 (SPSS Inc., Chicago, IL) was used for statistical analysis. The data were shown as the mean±standard deviation (SD). One-way ANOVA and LSD-t test were employed to compare between-group comparisons. It was considered to be significant different when P<0.05.