Formalin-fixed, paraffin-embedded (FFPE) bladder 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).
A total of 18 male patients with pathologically confirmed bladder cancer were enrolled in this study (Veterans Affairs Medical Center at San Francisco).
Three bladder cancer cell lines (J82; ATCC number: HTB-1, T24; ATCC number: HTB-4, UM-UC-3; ATCC number: CRL-1749) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The J82 and UM-UC-3 cell lines were cultured in MEM Eagle’s with Earle’s BSS supplemented with 10% fetal bovine serum. The T24 cell line was cultured in McCoy’s 5A medium with 10% fetal bovine serum.
RNA and Protein Extraction
RNA (microRNA and total RNA) was extracted from formalin-fixed, paraffin-embedded (FFPE) human bladder cancer and non-cancerous normal bladder tissues (urothelial cells) using a miRNeasy FFPE kit (Qiagen) after laser micro-dissection based on pathologists reviews. Total RNA was also extracted from bladder cancer cell lines using a miRNeasy mini kit (QIAGEN). Cells were lysed with RIPA buffer (Thermo Scientific, Rockford, IL) containing protease inhibitors (Sigma, St. Louis, MO). Protein quantification was done using a BCA protein assay kit (Thermo Scientific, Rockford, IL). The NE-PER Nuclear and Cytoplasmic Extraction Reagent was used to extract nuclear and cytoplasmic protein fractions from bladder cancer cells (Thermo Fisher Scientific, Rockford, IL).
MicroRNA Transfection (pre-miR Precursor and miR Inhibitor)
Pre-miR™ miRNA precursors [negative control (miR-NC) or hsa-miR-182-5p, Ambion] were transiently transfected into bladder cancer cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Anti-miR™ miRNA inhibitor [negative control (inh-NC) or miR-182-5p inhibitor (miR-182 inhibitor), Ambion] were transiently transfected into bladder cancer cells by siPORT NeoFX Transfection Agent (Ambion) according to the manufacturer’s instructions. After transfection, cells were incubated at 37°C for 48 hours until assessment.
Cell Viability, Cell Invasion, Wound Healing Assay
Cell viability was measured 3 days after transfection with MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega). Data are the mean ± S.D. of 6 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 were re-suspended in culture medium without FBS and placed in the upper chamber in triplicate. After 48 hours incubation at 37o
C (5% CO2), cells migrating through the membrane were stained. The results were expressed as invaded cells quantified at OD 560nm. The wound healing process begins with tissue matrix remodeling, migration, and eventual closing of the wound area. Therefore this assay is frequently used for assessment of cancer cell migration. 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 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)].
Cells (48 hours after transfection) were washed twice with 1xPBS and trypsinized. After inactivating trypsin in complete medium, the cells were re-suspended in ice-cold 1x binding buffer (70 µl). Annexin V-FITC solution (10 µl) and 7-AAD viability dye (20 µl) were added to 70 µl of the cell suspensions. After incubation for 15 minutes in the dark, 400 µl of ice-cold 1x binding buffer was added. The apoptotic distribution of the cells in each sample was then determined using a FACS (Cell Lab QUANTA SC, Beckman Coulter, Fullerton, CA). Data are the mean ± S.D. of four independent experiments.
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 target genes mRNA 3′UTR 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. For miR-182-5p precursor transfection, bladder 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 their transfection).
Primer sequences used for plasmid construction.
Overexpression Plasmid of Target Genes (RECK, Smad4) and Functional Analyses
In order to construct target gene (RECK, Smad4) 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-Smad4 was obtained by subcloning a NheI–XhoI fragment from pTargeT-RECK/Smad4 into the NheI–XhoI site of pCMV6-Entry Vector.
Initially we transfected pCMV6-empty and pCMV6-RECK or -Smad4 into bladder cancer cells and RNA and protein were extracted. Overexpression of RECK or Smad4 was confirmed by real time RT-PCR and Western Blot analysis and functional analyses were performed.
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. Human GAPDH and RNU48 were used as an 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 (Thermo Scientific, Rockford, IL) 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 overnight at 4 4°C with primary polyclonal and monoclonal antibody against Smad4 (#9515), RECK (#3433), beta-catenin (#9562), CREB (#9197) and beta-tubulin (#2128) from Cell Signaling Technology, Beverly, MA. 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 or CREB levels.
All statistical analyses were performed using StatView (version 5; SAS Institute Inc., NC). A p-value of <0.05 was regarded as statistically significant.