GRP78 Participates in PCA3-regulated Prostate Cancer Progression

Materials and Methods

Cell culture. PC3 prostate cancer cell lines were obtained from ATCC (Rockville, MD, USA) and cultured in RPMI-1640 medium (Gibco, Grand island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) at 37°C in 5% CO₂ humidified incubator. Cells were harvested in logarithmic phase of growth for all experiments described below.

Plasmid construction and transfection. PCA3 overexpression vector pcDNA3.1(+)-PCA3 was constructed by amplifying PCA3 with primers 5’-AGATTTGTGTGGCTGCAGC-3’ and 5’-TCCTGCCCATCCTTTAAGG-3’ using a synthesized PCA3 sequence (GenBank number NR_015342.2) as template, which was inserted into the pcDNA3.1(+) vector. For plasmid transfection, cells were seeded in 6-well plate and cultured overnight to reach 70-80% confluency. Recombinant plasmid or empty vector was transfected into cells using X-treme GENE HP DNA transfection reagent (Roche, Indianapolis, IN, USA), according to the manufacturer's instructions. Cells were examined 48 h after transfection under an inverted fluorescence microscope to assess transfection efficiency. Cells were then harvested and subjected to following analyses.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was extracted from cells using RNAiso Plus reagent (TaKaRa, Dalian, China), according to the manufacturer's instructions. The concentration of RNA was determined by spectrophotometry (Nanodrop 2000/2000C; Thermo Scientific, Shanghai, China). Then, 2 μg total RNA was reverse transcribed using ReverTra Ace (TOYOBO, Shanghai, China). qRT-PCR analysis was performed on a Roter-Gene Q (QIAGEN, Hilden, Germany) with the Platinum SYBR Green qPCR SuperMix-UDG kit (Life Technologies Corporation, Carlsbad, CA, USA), according to the manufacturer's protocol. Cycling conditions were: 50°C for 2 min; 95°C for 5 min; followed by 40 cycles of 95°C, 10 s and 60°C, 45 s. Melting curve analysis and agarose gel electrophoresis were used to confirm the specific PCR products. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as endogenous reference.

Cell proliferation assay. Cells were seeded into 96-well plates at a density of 5×10³ cells/well and cultured overnight. Cell proliferation was measured using the CCK-8 assay (Biyuntian, Shanghai, China) in 24 h increments for up to 96 h. In brief, 10 μl CCK-8 solution was added into each well of cell culture and the plates were further incubated for another 2.5 h. Optical density was then measured by FACS analysis at the absorbance of 450 nm. Experiments were performed in triplicate and repeated at least three times independently.

Wound healing assay. When transfected cells reached 95% confluence in 6-well plates after transfection, a 200 μl pipette tip was used to draw a straight wound with the same width on the cell monolayer. After washing with phosphate buffered saline (PBS) and removing cell debris, the cells were cultured using a serum-free medium under standard conditions. The wound was observed under a light microscope (Olympus Corporation, Tokyo, Japan) at 40× magnification at two preselected time points (0 and 24 h). The widths of the wounds in each well were measured.

Migration and invasion assays. Cell migration and invasion were determined using a transwell chamber (8 μm pore size) with and without BD Matrigel (BD Biosciences, San Jose, CA, USA). The upper side of the membrane was coated with Matrigel for the invasion assay. After 24 h of transfection, a total of 1×10⁵ cells in 200 μl serum-free medium were plated to the upper chambers and 600 μl medium containing 10% FBS was used as a chemoattractant in the lower chambers. After 48 h, the cells on the upper side of the membrane were removed using cotton swabs, whereas the invaded cells on the lower side of the membrane were fixed, stained and counted using an inverted microscope at 400× magnification.

Annexin V fluorescein isothiocyanate (FITC)/propidum iodide (PI) double staining. An Annexin V FITC Apoptosis Detection kit (BD Biosciences) was used to assess the apoptosis of transfected cells. PC3 cells, which were transfected for 48 h, were collected and washed with PBS. Subsequently, the cells were resuspended in 1X Binding Buffer at a density of 1×10⁶ cells/ml. In total, 5 μl Annexin V FITC and 5 μl PI were added to the cells, which were incubated at room temperature for 15 min. Subsequently, the cells were analyzed by flow cytometry, using BD FACSDiva™ software v6.1.3 and BD CellQuest™ Pro (BD Biosciences).

Gel electrophoresis, mass spectrometry and protein identification. The procedure of 2D-polyacrylamide gel electrophoresis (2D-PAGE) was performed as described previously (15). All gels were stained with Coomassie brilliant blue R-350 (Amersham Biosciences, Uppsala, Sweden). Gels were made in triplicate to confirm the spot patterns and scanned with a Z320 scanner (Founder, Beijing, China). Gel images were analysed with Imagemaster software version 6.0 (GE Healthcare, Piscataway, NJ, USA). For quantification, each gel was normalized by the volume of its each spot divided by the total volume of all spots. Quantitative analysis was performed by Student's t-test. Protein spots were excised from gels and destained with 25 mM NH₄HCO₃ 50% (v/v) acrylonitrile. After drying, 10 ng trypsin in 10 ml of 25 mM NH4HCO3 was added for digestion for 12 h at 37°C. Peptides were analysed on a 5800 MALDI-TOF/TOF MS (Applied Biosystems/MDS SCIEX, Framingham, MA, USA). Mass spectrometry data were analysed automatically by GPS Explorer software (version 3.6; Applied Biosystems) integrated with Mascot version 2.1 (Matrix Science, Boston, MA, USA) against the SWISS-PROT protein database (SwissProt Release 55.0, 356194 sequences, 127836513 residues) for Homo sapiens. For tandem mass spectrometry (MS/MS) database searches, the mass tolerance of precursor ions and fragment ions was set to 150 ppm and 0.4 Da. The MS/MS analysis score was >60 (p<0.05). If one spot corresponded to more than one protein, the top-ranked protein in each spot was singled out from the multi protein family.

Protein extraction and western blotting. Total cellular protein was extracted from transfected cells and protein concentration was determined using a BCA protein assay kit (Beyotime, Beijing, China). These protein samples were resolved in 10% SDS denatured polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes with a pore size of 0.45 μm (Millipore, Billerica, MA, USA). After blocking in 5% skim milk in Tris-based saline-Tween 20 (TBST) for 60 min at room temperature, membranes were incubated with rabbit anti-hGRP78 antibodies at a dilution of 1:1,000 (all antibodies from Abcam, Cambridge, MA, USA) overnight at 4°C. After washed in TBST, the membranes were further incubated with a secondary goat anti-rabbit (1:5,000) antibody for 1 h. Enhanced chemiluminescence (ECL) solution was added onto the membranes and protein expression was quantified using The Laboratory Work Image Acquisition and Analysis Software (UVP, Upland, CA, USA). Actin was used as a loading control.

Immunohistochemical staining. Formalin-fixed tissue samples were embedded in paraffin and sliced into 4 μm sections. Immunohistochemical staining was performed as previously described (16). Briefly, the tumor tissue sections were deparaffinized, rehydrated and stained with anti-hGRP78 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and biotin-labeled secondary antibody (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), followed by incubation with diaminobenzidine (Zhongshan Golden Bridge Biotechnology Co., Ltd.) and counterstained with hematoxylin. Slides were dehydrated with various concentrations of ethanol and soaked in xylene and then mounted with neutral balsam and visualized using a light microscope (DM LB2; Leica, Nussloch, Germany).

Statistical analyses. The results are expressed as mean±standard deviation. All statistical analyses were performed using the SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Data were analyzed using a two-tailed Student's t-test or one-way analysis of variance. p<0.05 was considered statistically significant.