The HEK293 and the human prostate cancer cell lines PC-3, DU-145 and NCaP, were obtained from the American Type Culture Collection (Manassas, VA). The nontumorigenic benign human prostatic epithelial cells BPH-1, (derived from human prostate epithelium of benign pathology) was generously provided by Dr. Simon W. Hayward (Department of Urological Surgery, Vanderbilt University Medical Center). Cells are maintained in RPMI-1640 medium (Gibco™, Grand Island, NY), supplemented with 10% fetal calf serum (CSS), 100U penicillin and 100-mg/ml streptomycin, at 5% CO2 incubator at 37 °C.
Western Blot Analysis
Confluent cell cultures (80%) were washed with PBS, scraped, and cell pellets were harvested. Cells were disrupted with RIPA buffer (50 mM Tris-HCl, pH7.4, 1% NP40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/mL each of aprotinin, leupeptin, pepstatin, and 1 mM Na3VO4). Cell lysates were centrifuged at 5,000×g (15mins), resolved by SDS-PAGE, and transferred to Immobilon-P membranes (Millipore, Bedford, Mass.). Upon incubation with the primary and secondary antibodies, immunoreactive bands were detected using a chemiluminescent approach with the ECL kit (Pierce, Rockford, IL). Membrane fractions were prepared using the protein isolation kit (Pierce). Monoclonal antibodies against EMMPRIN, ZO-2, actin and tubulin were purchased from Santa Cruz Biotech (Santa Cruz, CA). Monoclonal antibodies against ZO-1 and AF6 were obtained from Invitrogen Zymed (San Francisco, CA) and BD Transduction (Lexington, KY), respectively.
Total RNA was extracted from cells using an RNAeasy kit (Qiagen, Valencia, CA). RNA samples (0.25µg) were subjected to reverse transcription (RT) PCR reaction in a 20-µl volume with poly-oligoT primer. The resulting cDNA was subjected to PCR using EMMPRIN specific primers. The first set of primers started with exon 1 and ended at exon 11: EF2 (5’-ATG GCG GCT GCG CTG TTC GTG-3’) and ER11 (5’-GGA GCA GGG AGC GTC CTC GGG-3’). The second set of primers started with exon 2 and ended at exon 11: EF1 (5’-ATG AAG CAG TCG GAC GCG TCT C-3’) and ER11. GAPDH primers (5’-CAG CAA TGC ATC CTG CAC-3’ and 5’-GAG TTG CTG TTG AAG TCA CAG G-3’) were used as control in the same PCR reactions. Thirty cycles of PCR reactions were performed and each cycle included 45 sec, 94°C; 45sec, 55°C; and 45sec, 72°C. The PCR products were analyzed on a 1.2% agarose gel. Amplicons are purified, cloned and sequenced by IDT (Coralville, IA).
shRNA Plasmid Construction and Transfection
Short hairpin RNA (shRNA) interference oligos, were designed using OligoEngine software (Seattle, WA) to specifically target EMMPRIN (NM_198589). Three oligos that target EMMPRIN (variant 2 mRNA) at nucleotides 98–116 (TGGCTCCAAGATACTCCTC), 277–295 (CCATGGGCACGGCCAACAT) and 776–794 (AGGCAAGAACGTCCGCCAG), are named as 98i, 277i and 776i, respectively. A scramble shRNA (TTCTCCGAACGTGTCACGT) was used as control. The oligos are cloned to pSUPER (neo + GFP) plasmid from OligoEngine according to the manufacturer’s instruction. Plasmids were amplified in DH5α cell and confirmed by sequencing.
Subconfluent cell populations were used for transfection using the FuGENE system (Roche, Indianapolis, IN). Briefly, the plasmid and Fugene reagent were combined and incubated for 20–30mins at room temperature. After transfection (36hrs), cells were subjected to cell sorting based on GFP expression and GFP positive cells were subsequently subjected to Western blotting. Stable tansfectants were cloned under Geneticin selection (Invitrogen) (300µg/ml), the generated clones were maintained in RPMI 1640 medium (150µg/ml Geneticin).
Cell Viability Assay
The MTT assay (based on the ability of viable mitochondria to convert MTT, a soluble tetrazolium salt, into an insoluble formazan precipitate) was used to assess cell viability. Cells were seeded into 96-well plates (2,500 cells/well) and incubated in growth medium (18–24hrs). After incubation with the MTT solution for 4 hrs, absorbance was read at A570 and the colorimetric reaction product was quantitated spectrophotometrically (BioTek, PowerWave XS, Winooski, VT).
Evaluation of Cell Cycle and Apoptosis
BrdU/PI (Bromodeoxy uridine and propidium iodide) method was used for the analysis of cell cycle progression and apoptosis. Cells (1×106/ml) were incubated with BrdU (20mM) (60min at 37°C), suspended in PBS, and fixed with ice-cold 95% (v/v) ethanol. Fixed cells were subsequently permeabilized using pepsin (0.04% w/v, 0.4mg/mL in 0.1N HCl). BrdU was probed with FITC labeled anti-BrdU (BD, San Jose, CA). Apoptosis among the different cell populations was evaluated using the terminal UTP end-labeling (TUNEL) technique. (Leica, Germany).
Cell Adhesion Assay
The ability of cells to attach to key ECM components, (fibronectin and laminin) was tested using fibronectin or laminin-coated 6-well multiwell plates (BD Bioscience). Prostate cancer epithelial cells were plated (105/well), and incubated at 37°C for 30mins, prior to fixing with methanol, and washed with PBS. Cells were counted from three random fields/well.
Evaluation of Cell Migration and Invasion
Confluent monolayer cells were wounded by scraping. Cultures were washed twice with medium, and then incubated at 37 °C for 16hrs to allow migration toward the gap. The number of migrating cells was determined under the microscope. The invasion potential of prostate cancer cells was assessed using Biocoat Matrigel invasion chambers (Becton Dickinson). Briefly, cells (5×104) resuspended in RPMI1640-based medium were added (250µl) into the invasion chambers and chambers were subsequently inserted into 24-well plates. Stained cells were photographed and counted.
Cells were plated on fibronectin-coated glass coverslips and fixed with 4% paraformaldehyde. Cells were permeablized in 0.1% (v/v) Triton-X 100 and were subsequently stained with rhodamine-phalloidin (Jackson ImmunoResearch, West Grove, PA). After rinsing with PBS (3×), slides were mounted with Vectorshield (Vector Lab, Burlingame, CA). Slides were examined under a laser-scanning confocal microscope (Leica Lasertechnik, Heidelberg, Germany).
Cell Aggregation Assay
Cells aggregation assay was performed as previously described (27
). Briefly, cells were suspended into single cells and dissociated cells were allowed to associate in medium (1hr) in 5% CO2
at 37°C, with gentle rotation of the plates. The number of cell aggregates in the parental control PC-3 and EMMPRIN shRNA transfectant cells was counted.
Data are expressed as Mean ± SD. Mann-Whitney and Student’s t tests were used to comparatively analyze the differences between groups in the various experiments.