Tissue samples and cell culture
Twenty-seven formalin-fixed, paraffin-embedded (FFPE) prostate cancer samples and 25 FFPE benign prostatic hyperplasia (BPH) samples were obtained from the Veterans Affairs Medical Center (San Francisco, CA). Informed consent was obtained from all patients. All slides were reviewed by a board certified pathologist for the identification of prostate cancer foci as well as adjacent normal glandular epithelium. The prostate cancer cell lines LNCaP, DU145, and PC3 were obtained from the American Type Culture Collection (Manassas, VA). The prostate cancer cell lines were grown in RPMI 1640 media (UCSF facility) supplemented with 10% fetal bovine serum (Atlanta biological, Lawrenceville, GA) and 1% penicillin/streptomycin (UCSF facility) and were maintained in an incubator with humidified atmosphere of 95% air and 5% CO2 at 37°C.
The contamination with mycoplasma in cells was tested by polymerase chain reaction (PCR) with e-Myco Plus Mycoplasma PCR Detection Kit (Boca Scientific, Boca Raton, FL). Genomic DNAs (50 ng) of cell lines were added to the tubes containing 1× PCR buffer, deoxynucleoside triphosphates, mycoplasma primers and Taq
DNA polymerase. PCR was processed according to the procedure recommended by manufacturers. The mycoplasma primers can amplify all important species of mycoplasma including Mycoplasma opalescens
, Mycoplasma arginini
, Mycoplasma fermentans
, Mycoplasma caviae
, Mycoplasma hyorhinis
, Mycoplasma orale
etc. To reduce the chance of false negative, all tubes contain artificial tumor necrosis factor-α gene and its primers as internal control. To test the integrity of DNA samples, a primer set to amplify a housekeeping gene was added to all tubes as sample control. DNA from mycoplasma-contaminated cell line K562 was added to a tube as a positive control. No mycoplasma was contaminated in prostate cancer cell lines PC-3, LNCaP and Du145, kidney cancer cell lines CAKI-2 and A498 and colorectal cancer cell lines C, Colo320, Caco2, HT29, LS174T, RKO and SW48 (supplementary Figure 2
is available at Carcinogenesis
Transfection and apoptosis assay
Cells were placed in six-well plates at 70–90% confluency 24 h before transfection in serum free media. Cells were transfected with various plasmids (2 μg/well) by using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacture’s protocol. After 72 h, cells were collected by trypsinization, washed with phosphate-buffered saline and dual stained with the viability dye 7-amino-actinomycin D and Annexin V- fluorescein isothiocyanate using Annexin V- fluorescein isothiocyanate /7-amino-actinomyicin D kit (Beckman Coulter, Brea, CA) according to the manufacturer’s protocol. Stained cells were immediately analyzed using a flow cytometer (Cell Lab Quanta SC; Beckman Coulter).
RNA and DNA extraction
Total RNA was extracted from microdissected FFPE tissue and cultured cells using a miRNeasy FFPE Kit (Qiagen, Valencia, CA) for FFPE tissues and RNeasy mini kit (Qiagen) for cultured cells. Total DNA was extracted from 21 microdissected FFPE prostate cancer tissues and adjacent normal tissues.
Cells were plated in six-well plates on Day 0, and treated with a final concentration of 5 μM 5-aza-2′-deoxycytidine (Sigma, St Louis, MO) daily beginning from Day 1, until Day 4. The treated cells were harvested on Day 5.
Quantitative real-time PCR
Complementary DNAs were synthesized by using specific-miRNA primers (Applied Biosystems, Foster City, CA). Gene expression levels were measured by real-time quantitative PCR using an Applied Biosystems 7500 Fast Sequence Detection and gene-specific Taqman assay kits. RNU48 was used as endogenous control to normalize expression data. The thermal cycling conditions were according to the TaqMan Fast Universal PCR protocol. Each sample was analyzed in quadruplicate.
Bisulfite modified DNA sequencing
Methylation status of CpG sites in the miR-145 promoter was determined by NaHSO3-sequencing method. DNA was treated with NaHSO3 and amplified by PCR with the following primer set: forward, 5′GTGTAGATAGTAGAGGGTAGTTT; reverse, 5′TCCCACATCCAACCTCACAAA. This PCR product, which covers the miR-145 promoter region from −224 to +194, was sequenced on an ABI sequencer with dye terminators (Applied Biosystems). The quotient of C over C + T at each CpG site indicates the percentage of methylation.
The bisulfite-treated genomic DNA was amplified by PCR with a set of primers for the unmethylated reaction and with another set of primers for the methylated reaction: (a) unmethylated forward primer (5′GGGTTTTTGGTATTTTTTAGGGTAATTGAAGTTTT) and reverse primer (5′AACCAAAATAAAATACCACACATCACCA); (b) methylated forward primer (5′GGGTTTTCGGTATTTTTTAGGGTAATTGAAGTTTC) and reverse primer (5′TAAAATACCACACGTCGCCG). PCR was performed at 95°C for 5 min, 40 cycles at 94°C for 30 s, 56°C (unmethylated primer) or 64°C (methylated primer) for 30 s and 72°C for 30 s followed by a final extension at 72°C for 10 min in a PTC-100 thermal cycler (MJ Research, Waltham, MA). The PCR products were loaded on 2.0% agarose gel for analysis.
Sequence analysis of the p53 gene
DNA was amplified by PCR using five pairs of primers corresponding to sequences of exons 4–8 of p53 genes (4forward, 5′AAGCTCCCAGAATGCCA and 4reverse, 5′TGAAGTCTCATGGAAGCCAG; 5forward, 5′GTGCCCTGACTTTCAACTCT and 5reverse, 5′CAACCAGCCCTGTCGTCTCT; 6forward, 5′GCCTCTGATTCCTCACTGAT and 6reverse, 5′CCAGAGACCCCAGTTGCAAA; 7forward, 5′CAAGGCGCACTGGCCTCAT and 7reverse, 5′CAGTGTGCAGGGTGGCAAGT and 8forward, 5′GACCTGATTTCCTTACTGCCT and 8reverse, 5′GAATCTGAGGCATAACTGCAC). The PCR product was sequenced on an ABI sequencer with dye terminators (Applied Biosystems).
Electrophoretic mobility shift assay
A DNA probe containing the p53 response element at 5′ upstream site (−829 to −810) of miR-145 was obtained by annealing two oligos. A DNA probe containing consensus p53 response element (5′ GCCGAGACATGCCTAGACATGCCTCAA) was obtained by annealing two oligos. EMSA was performed using an EMSA kit (Molecular Probes; Invitrogen) according to the manufacturer’s instructions.
Laser captured microdissection (LCM) and RNA extraction from FFPE human prostate cancer samples
Microdissection was performed using the AutoPix System from Arcturus. Sections (8 μm) were placed on glass slides, deparaffinized, stained with hematoxylin, dehydrated and placed in the AutoPix instrument for microdissection. Areas of interest were captured with infrared laser pulses onto CapSure Macro LCM Caps ().
Fig. 1. Laser capture microdissection (LCM) in prostate tissues. Upper and lower panels are two prostate tissues. The left panels show targeted glands and stroma before LCM. The center panels show gland cells captured by LCM. The right panels show residual tissues (more ...)
In situ hybridization
Non-radioactive in situ hybridization was performed on 5 μm sections from FFPE prostate cancer tissue blocks using the digoxigenin-labeled locked nucleic acid modified detection probe (20 μM; 5′-AGGGATTCCTGGGAAAACTGGAC-3′, Exiqon, Tustin, CA) complementary to mature miR-145 and IsHyb in situ hybridization Kit (BioChain, Hayward, CA) following the protocol from Exiqon. Briefly, 5 μm sections were deparaffinized, hydrated, treated with proteinase K (10 μg/ml; Qiagen) at 37°C for 5 min then with 0.2% glycine for 1 min and fixed with 4% paraformaldehyde for 10 min. The sections were incubated in pre-hybridization solution at 50°C for 3 h and then in hybridization solution with digoxigenin -labeled, locked nucleic acid-modified miR145 probe at 57°C for 16 h. The sections were incubated in the phosphate-buffered saline diluted anti- digoxigenin -AP antibody (1:200) at 4°C for 16 h, washed with 2× standard saline citrate, 1.5× standard saline citrate, 0.2× standard saline citrate, incubated in blocking solution for 1 h at room temperature, washed with phosphate-buffered saline three times, then washed with 1× alkaline phosphatase buffer twice. BM purple (Roche South San Francisco, CA) was used for visualization of hybridization signal.
Statistical analysis was performed with Statview 5.0 for Windows as needed. Wilcoxon signed-rank test was used to compare the matched prostate cancer and adjacent normal samples. The student’s t-test was used to compare the different groups. P values <0.05 were regarded as statistically significant and are represented by an asterisk on the bars in the figures.