The design and schematic representation of this project
Schematic representation of the role of beta-catenin and MEK-ERK cascades in BC is shown in . The aim of this project was to identify new therapeutic miRNAs related to beta-catenin and MEK-ERK cascade pathways in BC cells. In order to identify miRNAs targeting these oncogenes, we initially used miRDB (25
) and microRNA.org
and found several miRNAs targeting them. The miRNAs identified are shown in .
Fig. 1. Role of beta-catenin and the Ras-Raf-MEK-ERK cascade in BC cells and potential miRNAs targeting beta-catenin and MEK-ERK. (A) Signal transduction of beta-catenin and the MEK-ERK cascade (B) Based on miRDB and microRNA.org, several miRNAs targeting beta-catenin (more ...)
Tissue array samples
We purchased a human BC tissue array from US Biomax, Inc. (catalog#: BL801; Rockville, MD) to detect miR-1826 localization and confirm miR-1826 expression levels in BC tissues by ISH. Tissue array patient information is shown in Supplementary Table S1
(available at Carcinogenesis
A total of 19 patients (all male) with pathologically confirmed BC were enrolled in this study (Veterans Affairs Medical Center at San Francisco). Samples were obtained from the patients after written informed consent.
A normal bladder epithelial cell line (SV-HUC-1; ATCC number; CRL-9520) and three BC cell lines (J82; ATCC number: HTB-1, T24; ATCC number: HTB-4, TCCSUP; ATCC number: HTB-5) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The SV-HUC-1 cells were cultured in ATCC-formulated F-12K Medium with 10% fetal bovine serum (FBS). The J82 cell line was cultured in minimum essential medium (Eagle) in Earle’s basic salt solution supplemented with 10% FBS. The T24 cell line was cultured in McCoy’s 5A medium with 10% FBS. The TCCSUP cell line was cultured in minimum essential medium (Eagle) in Earle’s basic salt solution supplemented with non-essential amino acids, 1 mM sodium pyruvate and 10% FBS.
RNA and protein extraction
RNA (miRNA and total RNA) was extracted from formalin-fixed, paraffin-embedded human BC and non-cancerous normal bladder tissues using a miRNeasy FFPE Kit (QIAGEN) after laser microdissection based on pathologist reviews. Total RNA was also extracted from renal cancer cell lines using a miRNeasy Mini Kit (QIAGEN). Cells were lysed with RIPA buffer (Pierce, Brebieres, France) containing protease inhibitors (Sigma, St. Louis, MO). Protein quantification was done using a BCA Protein Assay Kit (Pierce).
miRNA transfection (pre-miR precursor and miR inhibitor)
Pre-miR™ miRNA precursors [negative control (miR-NC) or hsa-miR-1826 (miR-1826); Ambion] were transiently transfected into BC cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Anti-miR™ miRNA inhibitor [negative control (inh-NC) or miR-1826 inhibitor (miR-1826 inhibitor); Ambion] were transiently transfected into BC cells by siPORT NeoFX Transfection Agent (Ambion) according to the manufacturer’s instructions. After transfection, cells were incubated at 37°C for 48 h until assessment.
Cell viability, cell invasion and wound healing assay
Cell viability was measured 4 days after miR-1826 transfection with MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI). Data are the mean ± SD of six independent experiments. Cell invasion assay was performed with the CytoSelect 24-well cell invasion assay kit as described previously (Cell BioLab, San Diego, CA) (26
). Transfected cells (miR-NC or miR-1826 transfectant—48 h) were resuspended in culture media without FBS and placed into the upper chamber in triplicate. After 48 h incubation at 37°C (5% CO2
), cells migrating through the membrane were stained and counted with a microscope. The results were expressed as invaded cells quantified at OD 560 nm. Cells undergoing apoptosis generally exhibit nuclear fragmentation and the apoptotic cells cannot invade the membrane. Therefore, cells migrating through the membrane do not include apoptotic cells. 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 as described previously (Cell BioLab) (26
). To generate a wound field, transfected cells (miR-NC or miR-1826 transfectants—48 h) were cultured until they formed a monolayer around the insert. After removing 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 at 10 h [percent closure rate (%) = migrated cell surface area/total surface area × 100)].
Apoptosis and cell cycle analyses
Cells (48 h after transfection) were washed twice with 1× phosphate-buffered saline (PBS) and trypsinized. After inactivating trypsin in complete medium, the cells were resuspended in ice-cold 1× 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 min in the dark, 400 μl of ice-cold 1× binding buffer was added. The apoptotic distribution of the cells in each sample was then determined using an FACS (Cell Lab QUANTA SC; Beckman Coulter, Fullerton, CA). The various phases of cells were determined using a DNA stain (4′,6-diamidino-2-phenylindole). Cell populations (G0/G1, S and G2/M) were measured using fluorescence and contrasted against cell volume. Data are the mean ± SD of four independent experiments.
3′ UTR luciferase assay
PmirGLO Dual-Luciferase miRNA Target Expression Vector was used for 3′ UTR luciferase assays (Promega). The primer sequences used were shown in Supplementary Table S2
(available at Carcinogenesis
Online). In a total volume of 20 μl, 5 μl of 100 μM forward primer, 5 μl of 100 μM reverse primer, 2 μl of 10× annealing buffer (100 mM Tris–HCl, pH 7.5; 1 M NaCl and 10 mM ethylenediaminetetraacetic acid) and 8 μl water were added to a 200 μl PCR tube, incubated at 95°C for 5 min and then placed at room temperature at 1 h. 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). The primers used were as follows: forward primer, 5′-cgtgctggaacacggtaaaa-3′; reverse primer, 5′-gcagccaactcagcttcctt-3′; PCR parameters for cycling were as follows: 94°C for 3 min, 30 cycles of PCR at 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, 72°C for 10 min and 4°C for 10 min. The PCR product was digested with NotI (TaKaRa/Fisher Scientific, Pittsburgh, PA). The size of the vectors containing inserts were about 200 bp and 100 bp by electrophoresis since the NotI recognition sequence was incorporated into the primers. Vectors were sequenced directly by an outside vendor (MCLAB, South San Francisco, CA). For 3′ UTR luciferase assay, BC cells were co-transfected with miR-NC and pmirGLO or miR-1826 and pmirGLO Dual-Luciferase miRNA Target Expression Vectors using Lipofectamine 2000 (Invitrogen). Luciferase assay was performed using the Dual-Luciferase® Reporter Assay System (Promega) at 48 h after transfection.
siRNA knockdown of CTNNB1 and MEK1 mRNAs and functional analyses
BC cells were transiently transfected with CTNNB1 or MEK1 siRNA (si-CTNNB1 and si-MAP2K1, catalog#VHS50819, #VHS40795; Invitrogen) or negative control siRNA (si-NC; Invitrogen) according to the manufacturer’s instructions. Briefly, cells were grown in six-well plates and transfected individually with si-NC (negative control) or si-CTNNB1 or si-MAP2K1 at a concentration of 200 pmol per well. Transfection was performed with X-tremeGene siRNA Transfection Reagent (Roche Diagnosis, Basel, Switzerland). Then, 48 h after transfection, RNA and protein were extracted, and knockdown of CTNNB1 and MAP2K1 mRNAs and proteins were confirmed by real-time RT–PCR and western blot analysis. Cell viability (24, 48 and 72 h after transfection), invasion (48 h after transfection) and apoptosis analysis (48 h after transfection) were performed using si-NC-transfected, si-CTNNB1-transfected or si-MAP2K1-transfected BC cells.
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). The TaqMan probes and primers were purchased from Applied Biosystems. Human GAPDH was used as an endogenous control and RNU48 was used as internal control. Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems).
Western blot analysis
Total cell protein (15 μg) was used for western blotting. Samples were resolved in 4–20% Precise Protein Gels (Pierce) and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Fairfield, CT). The membranes were immersed in 0.3% skim milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h and probed with primary polyclonal or monoclonal antibody against beta-catenin (#9562; Cell Signaling Technology, Beverly, MA), survivin (#2808; Cell Signaling Technology), MEK1 (#2352; Cell Signaling Technology), beta-Tubulin (#2128; Cell Signaling Technology) and VEGFC (#AP2042d; ABGENT, San Diego, CA) overnight at 4°C. Blots were washed in Tris-buffered saline containing 0.1% Tween 20 and labeled with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibody (Cell Signaling Technology). Proteins were enhanced by chemiluminescence (Amersham ECL plus Western Blotting detection system, Fairfield, CT) for visualization. The protein expression levels are expressed relative to beta-Tubulin levels.
Immunostaining of beta-catenin (CTNNB1), MEK1 (MAP2K1) and VEGFC was performed on formalin-fixed paraffin-embedded specimens using an antibody against human beta-catenin (#9562; Cell Signaling Technology), MEK1 (#ab47422; Abcam, Cambridge, MA) and VEGFC (#AP2042d; ABGENT). Immunohistochemical staining was evaluated by visually assessing staining intensity (0–2) using a microscope at ×200. All specimens were scored blindly by two observers. The intensity of beta-catenin expression in cells membrane was assessed. The criteria of intensity are as follows: 0, negative expression; 1+, weakly positive expression; 2+, strongly positive expression.
In situ hybridization
We performed ISH using BC tissue array (catalog#: BL801; US Biomax, Inc) and IsHyb In Situ Hybridization Kit (BioChain, Hayward, CA) in order to detect miR-1826 in human bladder tissues (normal and cancer) following the manufacturers’ protocol. Briefly, tissue array slides were incubated at 60°C for 1 h, deparaffinized and rehydration. Slide sections were fixed with 4% paraformaldehyde in Diethyl pyrocarbonate (DEPC)-PBS at room temperature for 20 min and washed with DEPC-PBS. Then, sections were treated with proteinase K (BioChain) for 15 min at 37°C, rinsed with DEPC-PBS, fixed again with 4% paraformaldehyde for 15 min and rinsed with DEPC-water. Slides were pre-hybridized at 50°C for 3 h with pre-hybridization buffer before an overnight 45°C incubation in hybridization buffers containing either 5′-DIG-labeled miR-1826 miRCURY™ LNA detection probe (Exiqon, Woburn, MA) or scrambled negative control probe (Exiqon) at 50 nM final concentration. Probe sequence is as follows: ATTGCGTTCGAAGTGTCGATGATCAAT. Slides were washed in 2× standard saline citrate (45°C for 10 min), 1.5× standard saline citrate (45°C for 10 min) and 0.2× standard saline citrate (37°C for 20 min). Slides were then exposed to 1× blocking solution (BioChain) for 3 h at room temperature. Then, slides were exposed to AP-conjugated anti-digoxigenin antibody for 1 h at room temperature. Then, slides were washed with 1× PBS for three times at room temperature (10 min per each) and again washed with 1× alkaline phosphatase buffer twice at room temperature (5 min per each). Then, slides were incubated with BM Purple AP substrate (Roche Diagnosis) in the dark for 2 h at room temperature. After rinsing, slides were counterstained in Methyl Green (BioVision, Mountain View, CA) and rinsed with Tris-buffered saline Tween-20 before mounting. A pathologist evaluated the ISH intensity of miR-1826 in bladder and non-BC cells using the same criteria that were used for immunohistochemistry.
All statistical analyses were performed using StatView (version 5; SAS Institute Inc., NC). A P-value of <0.05 was regarded as statistically significant.