Formalin-fixed, paraffin-embedded (FFPE) prostate cancer samples were obtained from the San Francisco Veterans Affairs (VA) Medical Center. Written informed consent was obtained from all patients and the study was approved by the UCSF Committee on Human Research (Approval number: H9058-35751-01).
All animal care was in accordance with the guidelines of the San Francisco Veterans Affairs Medical Center and the study was approved by the San Francisco VA IACUC (Protocol number: 08-003-01). Animal users have completed training programs to handle and work with mice through AALAS (American Association for Laboratory Animal Science) prior to animal experiments. A total of 8 nude mice (strain BALB/c nu/nu; Charles River Laboratories, Inc., Wilmington, MA) were used and initially prostate cancer cells were subcutaneously injected and tumor size was monitored as mentioned on page 7. After tumor growth, mice were euthanized with an overdose of isoflurane by inhalation. Then xenograft tissue was removed.
A total of 52 patients with pathologically confirmed prostate cancer were enrolled in this study (Veterans Affairs Medical Center at San Francisco). Samples were obtained from the patients after written informed consent. Detailed patient information is shown in .
Normal prostate epithelial cells (RWPE-1; ATCC number-CRL-11609) and prostate cancer cell lines (LNCaP; ATCC number-CRL-1740, PC-3; ATCC number-CRL-1435, DU145; ATCC number-HTB-81) were purchased from the American Type Culture Collection (Manassas, VA). The prostate cancer cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. RWPE-1 cells were cultured in keratinocyte-SFM (GIBCO/Invitrogen, Carlsbad, CA, USA). When purchased, permanent stocks of cells were prepared and all cells were stored at −80 degree until use. Cells were used for experiments within 6 months.
Total RNA and protein extraction
RNA (microRNA and total RNA) was extracted from formalin-fixed, paraffin-embedded (FFPE) human prostate cancer (n
52) and matched adjacent non-cancerous normal prostate tissues (n
43) or benign prostate hyperplasia (BPH) (n
9) using a miRNeasy FFPE kit (Qiagen) after laser capture micro-dissection based on a pathologist reviews. RNA (microRNA and total RNA) was also extracted from human cell lines using a miRNeasy mini kit (QIAGEN) and extracted from xenograft tissues homogenized in 1 ml TRIzol reagent (Invitrogen, Carlsbad, CA) then purified with RNeasy columns (QIAGEN). To digest DNA, a Qiagen RNase-Free DNase kit was used. Cells were lysed with RIPA buffer (Pierce, Brebieres, France) containing protease inhibitors (Sigma, St. Louis, MO). Protein was extracted from xenograft tissues using Tissue Protein Extraction Reagent (T-PER) (Thermo Scientific, Rockford, IL). Protein quantification was done using a BCA protein assay kit (Pierce, Brebieres, France).
MicroRNA and microRNA inhibitor transfection
Anti-miR™ miRNA inhibitor [negative control (miR-NC inhibitor) or miR-182-5p inhibitor (miR-182-5p inhibitor), Ambion] were transiently transfected into cancer cells with siPORT NeoFX Transfection Agent (Ambion) according to the manufacturer’s instructions. Pre-miR™ miRNA precursors [negative control (miR-NC) or hsa-miR-182-5p (miR-182-5p), Ambion] were transfected into cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Cell viability, cell invasion and wound healing assay
Cell viability was measured 3 days after transfection (miR-NC inhibitor/miR-182-5p inhibitor transfectant) with MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega). Data are the mean ± S.D. of 3 independent experiments. Cell invasion assays were performed with the CytoSelect 24-well cell invasion assay kit (Cell BioLab, San Diego, CA) according to the manufacturer’s instructions. Transfected cells (miR-NC inhibitor/miR-182-5p inhibitor transfectant-48 hours) were re-suspended in culture medium without FBS and placed in the upper chamber in triplicate. After 48 hours incubation at 37° C (5% CO2), cells migrating through the membrane were stained. The results were expressed as invaded cells quantified at OD 560 nm. Wound healing assay was performed with the CytoSelect 24-well wound healing assay kit according to the manufacturer’s instructions. To generate a wound field, transfected cells (miR-NC inhibitor or miR-182-5p inhibitor transfectant-48 hours transfection) were cultured until they formed a monolayer around the insert. After removing the insert, a 0.9 mm open wound field was generated and cells were allowed to migrate from either side of the gap. Wound closure was monitored and the percent closure was measured. [Percent closure rate (%)
migrated cell surface area/total surface area x100)].
Quantitative real-time RT-PCR
Quantitative real-time RT-PCR was performed in triplicate with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan universal PCR master mix according to the manufacture’s protocol (Applied Biosystems Inc., Foster City, CA, USA). The TaqMan probes and primers were purchased from Applied Biosystems. RNU48 was used as endogenous control. Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems).
Total cell protein (15–20 µg) was used for Western blotting. Samples were resolved in 4–20% Precise Protein Gels (Pierce, Brebieres, France) and transferred to PVDF membranes (Amersham Biosciences, Fairfield, CT). The membranes were immersed in 0.3% skim milk in TBS containing 0.1% Tween 20 for 1 hour and probed with primary polyclonal and monoclonal antibody against FOXF2 (#ab23306, Abcam, Cambridge, MA), RECK (#3433, Cell Signaling Technology), MTSS1 (#4385, Cell Signaling Technology), MMP−2 (#4022, Cell Signaling Technology) and beta-tubulin (#2128, Cell signaling Technology) overnight at 4°C. Blots were washed in TBS containing 0.1% Tween20 and labeled with horseradish peroxidase (HRP)-conjugated secondary anti-mouse or anti-rabbit antibody (Cell signaling Technology, Beverly, MA). Proteins were enhanced by chemiluminescence (Amersham ECL plus Western Blotting detection system, Fairfield, CT) for visualization. The protein expression levels were expressed relative to beta-tubulin levels.
Plasmid construction and 3’UTR-Luciferase assay
We constructed individual plasmids for each binding site in the 3’UTR of mRNA from potential target genes based on microRNA.org information. Then we confirmed miR-182-5p binding to the 3’UTR of target gene mRNA by luciferase assay with miR-182-5p precursor. PmirGLO Dual-Luciferase miRNA Target Expression Vector was used to perform 3’UTR luciferase assay (Promega, Madison, WI, USA). The primer sequences used for plasmid inserts are shown in . In a total volume of 20 µl, 5 µl each of 100 µM forward primer and reverse primer, 2 µl of 10x annealing buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM EDTA) and 8 µl water were added to a 200 µl PCR tube and incubated at 95 °C for 5 minutes then placed at room temperature for 1 hr. The oligonucleotides were ligated into the PmeI- XbaI site of pmirGLO Dual-Luciferase miRNA Target Expression Vector. Colony direct PCR was performed for insert recognition using REDTaq (Sigma, St. Louis, MO, USA). The primers used for PCR were as follows: forward primer, 5′-cgtgctggaacacggtaaaa-3′; reverse primer, 5′-gcagccaactcagcttcctt-3′. PCR parameters for cycling were as follows: 94°C for 3 minutes, 30 cycles of PCR at 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds, 72°C for 10 minutes and 4°C for 10 minutes. The PCR product was digested with NotI (TaKaRa/Fisher Scientific, Pittsburgh, PA, USA). The sizes of vectors containing inserts were about 200 bp and 100 bp by electrophoresis since the NotI recognition sequence was incorporated into the primers.
Primer sequences used for plasmid construction.
For miR-182-5p precursor transfection, prostate cancer cells were co-transfected with miR-NC and pmirGLO or miR-182-5p and pmirGLO Dual-Luciferase miRNA Target Expression Vectors using Lipofectamine 2000 (Invitrogen). Luciferase activity was assessed using the Dual-Luciferase® Reporter Assay System (Promega) (48 hours after transfection).
Establishment of stable low miR-182-5p expressing prostate cancer cells and effect on in vivo tumor growth
In order to observe the in vivo effect of miR-182-5p inhibitor on prostate cancer cells, we established stable low miR-182-5p expressing prostate cancer cell lines based on the lenti-viral system. We then used an in vivo nude mouse xenograft model. Lentivirus transfection was performed using a Lenti-Pac HIV Expression Packaging Kit (catalog #; HPK-LvTR-20, GeneCopoeia, Rockville, MD, USA) according to the manufacturer's instructions. Namely hsa-miR-182-5p inhibitor vector (catalog #; HmiR-AN0239-AM03, GeneCopoeia, Rockville, MD) or a miRNA inhibitor scrambled control clone pEZX-AM03 (catalog #; CmiR-AN0001-AM03, GeneCopoeia, Rockville, MD) with Lenti-Pac HIV mix were transfected into 293 Ta cells (GeneCopoeia) and incubated for 14 hours at 37°. The medium was then replaced with fresh medium containing 1/500 volume of the TiterBoost reagent. After 48 hours of incubation at 37°, the supernatant containing lentiviral particles was centrifuged at 500 x g for 10 min, filtered using 0.45 µm PES filter (Whatman/Fisher Scientific) and collected to sterile tubes. PC-3 cells were infected with lentiviral particles using Polybrene (8 µg/ml) (Sigma-Aldrich). After 24 hours incubation at 37°, medium was replaced with fresh medium to exclude Polybrene. Stably transfected cells (miR-182-5p low expressing or control) were selected using Hygromycin (100 µg/ml, Invitrogen, Carlsbad, CA, USA) for three weeks. Stable low miR-182-5p expressing prostate cancer cells (1×107/each) or control cells (1×107/each) were injected subcutaneously into the right back side flanks of 5 week-old nu/nu mice, respectively. A total of 8 nude mice (4-control, 4-miR-182-5p inhibitor) (strain BALB/c nu/nu; Charles River Laboratories, Inc., Wilmington, MA) were used for the in vivo xenograft model. MiR-182-5p expression was confirmed by real-time PCR when stable cell lines were injected into mice and when xenograft tissues were harvested. Tumor size was determined with calipers twice per week for 6 weeks, and tumor volume was calculated on the basis of width (x) and length (y): x2y/2, where x<y.
Overexpression of target genes (RECK, MTSS1) and functional analyses
In order to construct target gene (RECK, MTSS1) over expressing plasmids, the genes were amplified with total RNA from human adult normal kidney tissues (catalog#: R1234142-50, Biochain Institute, Newark, CA) and RWPE-1 by transcription–polymerase chain reaction (RT-PCR). The sequences of primers for cloning are shown in .
Polymerase chain reaction products were cloned into the pTargeT-Mammalian Expression Vector System (Promega, Madison, WI). Then pCMV6-RECK or pCMV6-MTSS1 was obtained by subcloning a NheI–XhoI fragment from pTargeT-RECK/MTSS1 into the NheI–XhoI site of pCMV6-Entry Vector. Initially we transfected pCMV6-empty and pCMV6-RECK or –MTSS1 into prostate cancer cells and RNA and protein were extracted. Overexpression of RECK or MTSS1 was confirmed by real time RT-PCR and Western Blot analysis and functional analyses were performed.
Effect of FOXF2 knock down on prostate cancer cell lines
We were not able to successfully make a FOXF2 over-expressing plasmid. Thus we performed functional analysis of FOXF2 using si-FOXF2 RNA in prostate cancer cell lines (PC3 and DU145). PC cells were transfected with FOXF2 si-RNA (Silencer Select si-FOXF2; Ambion) or negative control si-RNA [si-negative control (si-NC); Ambion] according to the manufacturer’s instructions. Briefly, cells were grown in six-well plates and transfected individually with the siRNA at a concentration of 200 pmol/well. The siRNA sequences are as follows: si-FOXF2- Sense; 5’-GAAAAGAUUUCGUCCUCAAtt-3’, Anti-sense; 5’-UUGAGGACGAAAUCUUUUCtg-3’. Transfection was performed with X-tremeGene siRNA Transfection Reagent (Roche Diagnosis, Basel, Switzerland). MTS, in vitro transwell invasion and wound healing assays were performed for functional analyses. For wound healing assay, after setting the wound, wound closure was assessed after 8 hours.
All statistical analyses were performed using StatView (version 5; SAS Institute Inc., NC). Error bars in all the figures represent S.D. (Standard Deviation). Statistical significance was determined using the Students t-test or Analysis of Variance (ANOVA) for functional analysis. Chi-square or the Fisher’s exact test was used for the association of clinical parameters with miR-182-5p expression. A p-value of <0.05 was regarded as statistically significant.