ATX (recombinant human ENPP-2/Autotaxin) and OPN antibody were purchased from R&D Systems (Minneapolis, MN, USA). Oleoyl-L-a-lysophosphatidic acid sodium salt (LPA), oleoyl-L-a-LPC, blasticidin, MAPK inhibitor (PD98059), PI3K inhibitor (LY294002), LPA receptor antagonist (Ki16425) and α-tubulin antibody (#T5168) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Monoclonal anti-p44/42 MAP kinase, anti-Phospho-p44/42 MAPK (Thr204/Tyr202), total-Akt and phospho-Akt (Ser473) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). pFR-Luc plasmid and pFA2-Elk1 plasmid were purchased from Invitrogen (Carlsbad, CA, USA).
Human gastric cancer SGC7901 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin sulfate (100 μg/ml), and maintained at 37°C with 5% CO2 in a humidified incubator.
Construction of OPN-siRNA expression plasmids
SiRNA against OPN was produced from Invitrogen (Carlsbad, CA, USA). The sequences of the selected region to be targeted by siRNA for OPN were:
We used lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) to separately transfect three types of OPN constructs into SGC7901 cells. To select resistant colonies, 48 hours after transfection, cells were cultured in selective medium containing 3 μg/ml blasticidin (Sigma-Aldrich, Saint Louis, MO, USA). Blasticidin-resistant cells were maintained in culture medium supplemented with 3 μg/ml blasticidin for further analysis.
Western blotting analysis
SGC7901 cells (1 × 106) were treated with ATX(50 ng/ml), LPC(10 μM), LPA(10 μM), ATX(50 ng/ml)+LPC(10 μM)(ATX/LPC1) or ATX(50 ng/ml)+LPC(20 μM) (ATX/LPC2) for 24 hours, and then cells were lysed in RIPA buffer. Equal amounts of protein (60 μg) were electrophoresed on 12% SDS-PAGE gels and electrophoretically transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). Membranes were incubated overnight at 4°C with anti-OPN (1:100) or monoclonal anti-α-tubulin (1:5000) in TBST containing 1% BSA (w/v). The membranes were incubated for 2 hours with anti-rabbit or anti-mouse secondary antibodies, and the immune complex was detected using an ECL plus detection kit (Pierce, Rockford, IL, USA). The optical densities of each band and the density ratio of OPN to α-tubulin bands were calculated using a densitometer (Furi, Shanghai, China).
Cell migration was performed using 24-well transwell migration plates (Corning Costar, Schiphol-Rijk, Netherland). The upper chamber was filled with 100 μl of cell suspension (1 × 105 cells) in DMEM/0.1% BSA. The lower chamber contained 600 μl of the DMEM/0.1% BSA with ATX (50 ng/ml), LPC (10 μM), LPA (10 μM), ATX/LPC2. The filters were fixed with methanol and stained with hematoxylin and eosin after 48h's incubation. Cells remaining on the top side of the filter were removed by soft mechanical scraping, and the number of cells migrating to the bottom of the filter was counted using a light microscope (in each chamber, six fields were counted at 200× magnification for each condition).
RNA extraction and Real-Time PCR
Total RNA was extracted from SGC7901 cells incubated in 6-cm plates after 18 hours of stimulation with either ATX (50 ng/ml), LPA (10 μM), LPC (10 μM), ATX/LPC1, ATX/LPC2, or DMEM/0.1% BSA alone as a negative control, using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. Reverse transcription was performed using a RevertAidTM First Stand cDNA Synthesis Kit (MBI-Fermentas). The transcript levels for OPN, LPA1, LPA2, LPA3, LPA4 and β-actin were quantified by real-time PCR. The primers used were as follows:
LPA4-R: 5-AAACAGGGACTCCAT TCT-3 ;
Flow cytometric analysis for apoptosis
SGC7901 cells were stimulated with the following treatments: Taxol (50 nM), or Taxol (50 nM) containing ATX (50 ng/ml), LPC (10 μM), LPA (10 μM), ATX/LPC2 for 24 hours. Cultured cells were collected with trypsin/EDTA and washed with PBS and stained with Propidium Iodide (PI) or FITC-conjugated antibodies. Fluorescence was quantified on 10,000 cells with FacsCalibur with Cellquest software (BD Biosciences, PharMingen)
Luciferase (LUC) reporter assay
SGC7901 cells (1 × 105) were transfected with pFR-Luc plasmid (reporter plasmid), or pFA2-Elk1 plasmid (fusion trans-activator plasmid). Six hours later, the cells were treated with the following drugs: ATX/LPC2, or ATX/LPC2 containing DMSO, Ki16425 (15 μM), PD98059 (50 μM), or LY294002 (50 μM) and incubated for an additional 24 hours. The cells were then harvested and tested using a Luciferase assay system (Promega, Madison, WI). An error bar was established to show the SD derived from three independent experiments.
Data were analyzed using a two-tailed Student's t-test for single comparisons and by one-way analysis of variance for multiple group comparisons. Differences were considered significant at * P < 0.05 versus control.