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).
Cell culture and transfection
Human prostate carcinoma cell lines, PC-3, LNCaP and DU145 and a non-malignant epithelial prostate cell line, RWPE-1, were purchased from The American Type Culture Collection (Manassas, VA). The prostate cancer cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). RWPE-1 cell line was cultured in keratinocyte growth medium supplemented with 5 ng/mL human recombinant epidermal growth factor and 0.05 mg/mL bovine pituitary extract (Invitrogen, Carlsbad, CA)
Cells in 6-well plates were transfected with 30 nM pre-miR negative control (NC) or pre-miR-34a (Applied Biosystems, Foster City, CA) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions.
Formalin-fixed, paraffin-embedded (FFPE) prostate cancer samples were obtained from the San Francisco Veterans Affairs Medical Center. All slides were reviewed by a board certified pathologist for the identification of prostate cancer foci as well as adjacent normal glandular epithelium.
Generation of stable miR-34a cell lines
An HIV-based lentiviral packaging system including a plasmid expressing miR-34a or vector control was purchased from GeneCopoeia (Rockville, MD). Lentivirus particles were produced by transfecting lentiviral expression plasmid into 293T cells. PC-3 cells were infected with the HIV-based lentivirus expressing miR-34a or vector control (1.5×106 infectious units of virus (IFU) (in 20 µl), and the infected PC-3 cells were selected with puromycin (0.5 ug/ml).
Soft agar colony formation assay
Soft agar colony formation assay was performed by seeding cells in a layer of 0.35% agar/RPMI/FBS over a layer of 0.5% agar/RPMI/FBS. RPMI/FBS was added every 5 days to continuously supply growth supplements to the cells. Cultures were maintained in a humidified 37°C incubator. On day 14 after seeding, colony forming efficiency was quantified by light microscopy.
In vivo tumor growth
Suspensions of the stable miR-34a expressing cells or the control cells (1×107 cells in 100 µl RPMI medium) were subcutaneously injected into female nude mice (strain BALB/c nu/nu; Charles River Laboratories, Inc., Wilmington, MA, 4–5 weeks old). Tumor volume was calculated on the basis of width (x) and length (y): x2y/2, where x<y.
RNA extraction and quantitative real-time PCR
RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Reverse transcription reactions were performed using a Reverse Transcription System Kit (Promega, Madison, WI). Quantitative real-time PCR analysis was performed in triplicate with an Applied Biosystems Prism7500 Fast Sequence Detection System using TaqMan universal PCR master mix according to the manufacturer's protocol (Applied Biosystems). Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems).
miRNA extraction and quantitative real-time PCR
Total RNA was extracted from laser capture microdissected (LCM) FFPE tissues and cultured cells using a miRNeasy FFPE kit (Qiagen, Valencia, CA) and an RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Reverse transcription reactions were performed using a Reverse Transcription System Kit (Applied Biosystems Inc., Foster City, CA). Quantitative real-time PCR analysis was performed as described above.
Putative target sites of the 3′UTR were cloned into the PmeI-XbaI site of the dual luciferase pmirGLO vector (Promega, Madison, WI). For mismatch constructs, 6 mismatches were introduced in the putative target site.
A human c-Myc expression vector was constructed by subcloning the full-length cDNA of c-Myc (Invitrogen, Carlsbad, CA) into the HindIII–XhoI site of the pCMV6-ENTRY vector (Origene).
c-Met expression vector (human ORF Myc-DDK-tagged ORF clone of Homo sapiens met proto-oncogene) was purchased from Origene (Rockville, MD).
Cell viability assay
Cell viability was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI), a colorimetric assay which measures the activity of reductase enzymes. Cells were seeded at a density of 1×103 cells per well in flat bottomed 96-well plates. At the indicated times, CellTiter 96 Aqueous One reagent was added to each well according to the manufacturer's instructions. Cell viability was determined by measuring the absorbance at 490 nm using a kinetic microplate reader (Spectra MAX 190; Molecular Devices Co., Sunnyvale, CA). Data are the mean ± standard deviation (SD) of 3 independent experiments.
Apoptosis was measured using flow cytometry (Cell Lab Quanta SC, Beckman Coulter, Brea, CA) with Annexin-V-FITC/7-AAD labeling. Measurements were repeated independently three times.
Transwell invasion assay
PC-3 cells were grown in DMEM containing 10% FBS. Culture inserts of 8-µm pore size (Transwell; Costar) were coated with Matrigel (BD Biosciences, San Jose) (100 µg per well) and placed into the wells of 24-well culture plates. In the lower chamber, 500 µl of DMEM containing 10% FBS was added and 1×105 cells were seeded to the upper chamber. After 48 hours of incubation at 37°C with 5% CO2, the number of cells that had migrated through the pores was fixed with 10% formalin and stained with 0.05% Crystal Violet. Crystal Violet was solubilized with methanol and absorbance (540 nm) of the solution was measured by a kinetic microplate reader (Spectra MAX 190; Molecular Devices Co., Sunnyvale, CA). Data are the mean ± S.D. of 3 independent experiments.
Protein extracts were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Hybond-P; GE Healthcare, Piscataway, NJ), followed by incubation with the indicated primary and secondary antibodies conjugated to horseradish peroxidise (GE Healthcare). Signals were detected using the ECL detection system (Amersham ECL plus Western Blotting detection system, Fairfield, CT). Antibodies against c-Myc, RhoA, Skp2 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against Miz1, Max, Cdk9, ARHGEF3 and ARHGEF18 were purchased from GeneTex (Irvine, CA). An antibody against CycT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Luciferase reporter assay
Cells in 24-well plates were transfected with 30 nM pre-miR negative control (NC) or pre-miR-34a (Applied Biosystems) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Transfections of plasmids were perfomed using FuGENE HD (Roche Diagnosis, Basel, Switzerland) according to the manufacturer's instructions. All transfection experiments were performed in triplicate. Luciferase activity was assayed at 48 h after transfection, using a dual-luciferase reporter assay system (Promega).
Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitations were performed using the Chromatin Immunoprecipitation Assay Kit (Epigentek, Brooklyn, NY) according to manufacturer's instructions. DNA was sheared by sonication. A 1% portion of the sheared DNA–protein complex was used for an input DNA sample. Antibodies against c-Myc or RNA polymerase II (Millipore, Billerica, MA) or normal rabbit IgG (Millipore) was used for immunoprecipitation. Real-time PCR quantitation of ChIP was performed in triplicate, normalized by input, and expressed as a fold increase over the control. Primers used for real-time PCR were as follows: RhoA E-box5/6, 5′ -CTTCGCGTGCGTGAAGAGTTG-3′ and 5′-CATCCACTATTGCTCAGGAGC-3′; GAPDH promoter, 5′-TACTAGCGGTTTTACGGGCG-3′ and 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′.
Cells were lysed in buffer containing 250 mM NaCl, 50 mM HEPES pH 7.5, 0.1% Nonidet P40, 5 mM EDT and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein A/G-Sepharose beads (Santa Cruz Biotechnology) were added to the lysate for 60 minutes at 4°C for preclearance. The precleared lysate was incubated with an anti-c-Myc antibody (Cell Signaling Technology) or normal rabbit IgG (Millipore) and protein A/G-Sepharose beads overnight at 4°C. The protein A/G-Sepharose beads were washed with the lysis buffer. The proteins were separated by SDS-PAGE and analyzed by Western blot.
Data are shown as mean values ± standard deviation (SD). The Student's t-test was used to compare the two different groups. P values of less than 0.05 were regarded as statistically significant (n