- Tissue samples
In the study, a total of 81 prostate cancer samples and 22 normal prostate samples were obtained from patients who underwent prostate biopsy in Department of Urology, Ningbo First Hospital (Ningbo, China) between 2012 and 2017. These samples were then formalin-fixed and paraffin-embedded followed by pathological diagnosis and immunohistochemical staining. The study protocol was approved by the Ethics Committee of Ningbo First Hospital and the information written consent was obtained from all the subjects prior to their participation in the study. The clinicopathological characteristics of patient samples were obtained from medical records within Ningbo First Hospital and were summarized in Table 1.
- Immunohistochemical staining
Immunohistochemical staining (IHC) was performed on paraffin-embedded tissue sections to determine PNN levels in prostate cancer tissues and normal prostate samples using a staining kit (absin, Shanghai, China). Briefly, sections were deparaffinized with xylene and rehydrated with ethanol-aqueous solutions, then antigen retrieval was done by heating the slides for 15 min in a microwave oven in 10 mM citrate buffer (pH 6.0). After eliminating endogenous peroxidase activity using 3% H2O2 and blocking with 5% BSA, the sections were incubated with anti-PNN antibody (1:50, HPA001378, Sigma-Aldrich, Merck KGaA, Germany) overnight at 4°C and followed with secondary antibody. The sections were then incubated with Diaminobenzidine (DAB) and counterstained with Mayer’s hematoxylin.
- TCGA data analysis
The gene expression matrix and clinical phenotype of prostate cancer patients from TCGA were downloaded from UCSC (https://xenabrowser.net/datapages/), then PNN expression in prostate cancer was analyzed by GraphPad Prism 8.0. Kaplan-Meier survival curves, applied to analyze the effect of PNN expression on PCa patients’ recurrence and survival, were analyzed with GEPIA (Gene Expression Profiling Interactive Analysis) [19].
- Cell culture and cell transfection
Human prostate cancer cells DU145, 22Rv1, LNCaP clone FGC and PC-3, and human embryonic kidney cells 293T used in the study were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in MEM, RPMI 1640, F-12K or DMEM medium (Gibco, Thermo Fisher SCIENTIFIC, USA) containing 10% fetal bovine serum (PAN, Germany). All cell cultures were carried out in a humidified chamber at 37 °C with an atmosphere of 5% CO2.
PNN expression vector pcDNA3.1-3xFlag-C-PNN (pcDNA3.1-PNN) and primer vector pcDNA3.1-3xFlag-C (pcDNA3.1) used in the study was purchased from YouBio (Changsha, Hunan, China). Lipofectamine™ 3000 reagent (Invitrogen, Thermo Fisher SCIENTIFIC, USA) was used to cell transfection following the manufacturer’s instructions.
- Lentivirus production and generation of stably PNN-knockdown PC-3 cells
The shRNAs targeted human PNN were obtained from GPP Web Portal (https://portals.broadinstitute.org/gpp/public/gene/search). The shRNAs sequences used in the study included shPNN #1 (5'-CCGACAGAAAGAGGTCTATAT-3'), shPNN #2 (5'-GAAGGTAGACGCATCGAATTT-3'), shPNN #3 (5'-GGTAGAGGACGTGGTAGTTTA-3') and a scramble shRNA (5'-CCTAAGGTTAAGTCGCCCTCG-3'). We cloned the shRNAs into pLKO.1-puro (Plasmid #8453, Addgene) at Age Ⅰ and EcoR Ⅰ to construct pLKO.1-shPNN or pLKO.1-shSCR vectors. Three plasmids pCMV-dR8.2 dvpr (Plasmid #8455, Addgene), pCMV-VSV-G (Plasmid #8454, Addgene) and pLKO.1-shPNN or pLKO.1-shSCR were co-transfected into 293T cells and cultured for 72 h. Lentivirus-containing supernatants were then harvested using 0.45 μm sterilizing filter. Lentiviruses were then used to infect PC-3 cells and the infected cells were then selected by complete medium containing 2ug/ml puromycin to acquire stably PNN-knockdown PC-3 cells.
- Western blotting
Tumor tissues and cells were lysed and protein was harvested with RIPA buffer (Solarbio, Beijing, China) supplemented with 1% protease inhibitor mix (Cell Signaling Technology, USA) and 1% phosphatase inhibitor mix (Sangon Biotech, Shanghai, China). Proteins were separated on SDS-PAGE gel, then transferred onto PVDF membranes (BIO-RAD, USA) followed with blocking by 5% non-fat milk. The membranes were incubated with the primary antibody at 4℃ overnight. The following primary antibodies were used: E-Cadherin, N-Cadherin, Vimentin, Fibronectin, GAPDH, MMP-2, MMP-9, CDK4, CDK6, CDK2, CyclinD1, CyclinE1, PI3K p110α, PI3K p110β, PI3K p110γ, PI3K p85, p-PI3K p85(Tyr458), AKT, p-AKT (Ser473), CREB and p-CREB (Ser133) were purchased from Cell Signaling Technology (USA), PNN antibody was obtained from Sigma-Aldrich (Merck KGaA, Germany). Then, membranes were incubated with HRP (horseradish peroxidase)-labeled secondary antibody (Boster, Wuhan, China) and detected by chemiluminescence.
- RNA extraction and quantitative real time-PCR (qRT-PCR)
Total RNAs were extracted from prostate cancer cells using TRIzol Reagent (Invitrogen, Thermo Fisher SCIENTIFIC, USA). The RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher SCIENTIFIC, USA) was used to synthesize cDNA. The cDNAs were amplified by qRT-PCR using SYBR Green PCR Master Mix (Roche, US) on a LightCycler480 system, and fold changes were calculated by relative quantification (2-ΔΔCt). The PCR primers sequences were as follows: Forward primer: 5'-ACCCACTCCTCCACCTTTGAC-3' and Reverse primer: 5'-TGTTGCTGTAGCCAAATTCGTT-3' for GAPDH, Forward primer: 5'-CCTGTAAAGCAGTCTCAAGCC-3' and Reverse primer: 5'-CGAATGTTCTCATCCACGTTCT-3' for PNN.
- Transwell assay and wound healing assay
The transwell assay was performed to determined the ability of cellular invasion and migration using 8 μm Transwell® Permeable Supports (Costar, Corning, Bedford, MA, USA). For cell migration assay, a suspension of cells (1 × 105 cells/well for PC-3 and 5 × 104 cells/well for DU145) in serum free basal medium was seeded to the upper chambers. The lower chambers were filled with growth media containing 10% FBS. After incubation at 37°C (24 h for DU145 cell and 48 h for PC-3 cell), the non-invaded cells in upper chamber were removed and the invaded cells were fixed with 90% methyl alcohol, then stained with 0.1% crystal violet (Sigma-Aldrich, Merck KGaA, Germany). The invasion assay was performed using the same procedure with the following modifications: (i) the seeding density was 2 × 105 cells/well for PC-3 and 1 × 105 cells/well for DU145, (ii) the upper chambers were pre-coated with Matrigel® Matrix (Corning, Bedford, MA, USA), and (ⅲ) PC-3 and DU145 cells were all incubated for 48 h at 37°C.
The wound healing assay was performed to determine the cell motility. When the seeded cells reached 95% confluence in 24-well plate, a linear wound was created with a micropipette tip across the diameter of the well, and then PBS was used to rinse the non-adherent cells. The medium containing 0.5% FBS was added to allow cells to move into the gap without the influence of cell growth. Three different equidistant points of the scratched area were photographically measured and imaged by an inverted phase contrast microscope (Olympus, Japan) at 0 h and 24 h. Migration rate was calculated as the proportion of initial scratch distant of each sample using the mean distance between both borderline that remain cell-free after cell migration.
- MTS assay
MTS assay was performed to determined cell proliferation using Cell Titer 96® Aqueous One Solution Reagent (MTS, Promega, Madison, USA) according to the manufacturer’s protocol. In brief, a suspension of cells (2000 cells/well for DU145 and 4000 cells/well for PC-3) in 100 μl of growth media was seeded to 96-well plate,
Following incubation for 4, 24, 48, 72 and 96 h, 20 μl of MTS reagent was added to each well. The absorbance (OD value) was measured at 490 nm after incubation for another 2 h using iMarkTM Microplate Reader (Bio-Rad, US).
- Cell cycle and apoptosis assay
Cells were harvested by trypsinization and washed with PBS, then the cells were stained followed the Cell Cycle Staining Kit (MultiSciences, Hangzhou, China). After staining, cell cycle analysis was performed using a flow cytometer (Beckman Coulter, Fullerton, CA, USA).
Cell apoptosis was assessed using Annexin V-FITC/PI apoptosis kit (MultiSciences, Hangzhou, China). In brief, cells were harvested and washed with pre-cold PBS, then cells were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and PI followed with apoptosis analysis by Beckman flow cytometer.
- Tumorigenesis assay of tumor cells
Colony formation assays and Xenograft model in nude mice were performed to detect the tumorigenicity of prostate cancer cells. For colony formation assays, PC-3 cells were counted and seeded in 6-well plate at a density of 1000 cells per well. After incubation for 14 days, the cells were fixed with 90% methyl alcohol for 15 min and then stained with 0.1% crystal violet (Sigma-Aldrich, Merck KGaA, Germany) for 15 min. The number of colonies consisting of more than 50 cells was counted.
Five-week-old male nude mice (BALB/C) (Shanghai laboratory animal center, China) were used as xenograft model with a protocol approved by the Institutional Animal Ethics Committee of Ningbo University. 4 × 106 stably PNN-knockdown PC-3 cells or PC-3/Control cells were injected subcutaneously into the flank of mouse (5 for each group) and tumor formation was monitored. On day 32 after inoculation, the nude mice were sacrificed to collect the tumors.The tumor volume was calculated with the formula V = L*W2/2 (V, volume; L, length; W, width).
- Statistical analysis
Statistical analyses were performed with SPSS software (SPSS Inc., Chicago, IL, USA). The correlation between PNN expression and the clinicopathological features was analyzed with chi-square test. Bivariate correlations between study variables were calculated using the Spearman's rank correlation coeffcient. Variance among the control and tested groups were analyzed using one-way ANOVA analysis followed by Dunnett post hoc test. Variance between two groups was assessed using student t test. The data were showed as Mean ± standard deviation (SD). P < 0.05 was considered statistically significant in all tests.