MicroRNAs
miR-618 may be a regulator of TIMP-1 molecule according to target prediction tools (http://www.targetscan.org). mir-618, anti-miR-618 and positive and negative controls (Ambion, Austin, TX, USA) were diluted to 10 μM stock solutions and stored frozen at − 20 °C until use. All experiments were performed in triplicate.
Cell lines
The DU145 cell line was used (American Type Culture Collection - ATCC). Cells were placed in medium containing DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution (Sigma Co., St. Louis, MO, USA). The plates were maintained at 37 °C, 95% air and 5% CO2.
Cell transfection
Transfections were performed with Lipofectamine (siPORT NeoFX -AMBION, USA) with the following protocol: The day before transfection, 6 × 104 cells were maintained without antibiotic. Approximately 2.5 μL of 10 μM solution was diluted in 50 mL of OPTI-MEM and mixed with a solution of 1.5 μL of transfection agent diluted in 50 mL of OPTI-MEM I. Then, 100 μL of transfection complex was dispensed on a 12-well culture plate and incubated for 24 h in CO2 at 37 °C.
Total RNA and miRNA extraction
Twenty-four hours after transfection, the cells were trypsinized and centrifuged at 4000 rpm for 5 min. Total RNA and miRNA were extracted with a mirVana kit (Applied Biosystems, Foster City, CA, USA). The purity and concentration of the miRNA and RNA were measured with a spectrophotometer (ND-1000, Thermo Scientific, Wilmington, USA) at a wavelength between 260 and 280 nm (A260/280).
Reverse transcription (RT)
Reverse transcription was performed using the TaqMan Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instructions. The synthesis of TIMP-1 cDNA was performed with 5 ng of mRNA (High-Capacity cDNA Reverse Transcription Kit - Applied Biosystems) using reverse transcriptase and random primers.
For miRNA, the reaction was performed with Veriti equipment (Applied Biosystems) with the following parameters: 30 min at 16 °C, 30 min at 42 °C and 5 min at 85 °C. The cDNA from RNA was obtained with same equipment using the following parameters: 10 min at 25 °C, 120 min at 37 °C and 5 min at 85 °C.
Real-time PCR
We used the ABI 7500 FAST thermocycler to assess the efficacy of transfection and the expression of the TIMP-1 and MMP-9 genes. The reactions were performed with 0.5 μL of specific primer, 5 μL of TaqMan® Universal PCR Master Mix (Applied Biosystems, California, USA), 3.5 μL of nuclease-free water and 1 μL of RNA cDNA. Primers employed in the study detected mature miRNAs. The PCR cycles were 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s 95 °C and 1 min at 60 °C. The endogenous control was B2M for the genes and RNU43 for the miRNAs. The expression levels of the miRNAs and target genes were determined, and the relative quantification of expression levels, expressed in fold changes, was determined by the 2-ΔΔct method [8].
Elisa
The ELISA (R&D Systems, Minneapolis, MN, USA) assay for MMP-9 detection was conducted with the conditioned medium from DU145 cells transfected with miR-618 or its antagonist according to the manufacturer’s instructions. A standard curve was prepared on each plate in duplicate. Samples were diluted two-fold for analysis. All analyses were performed in duplicate, and positive and negative controls were employed for statistical analysis.
Zymography
The conditioned medium from the top of the Matrigel membrane was collected, and total protein was quantified using a BCA Protein Assay kit (Thermo Scientific). Approximately 40 μL of sample was added to 4–10% polyacrylamide gel containing 0.1% gelatin. Ten microliters of the protein standard (Chemicon Co.) was also loaded onto each gel. Electrophoresis was then performed at 4 °C, and the gel was washed in Tris including 2.5% Triton X-100 (Bio-Rad#161–0407) to remove the SDS and allow protein renaturation. Then, the gels were incubated for 48 h in buffer solution gel (BIO-RAD 10x Zymogram #161–0766) and stained with Coomassie blue R-250. Revelation was performed after washing the gels with a solution of 40% methanol and 10% glacial acetic acid in distilled water. MMP-9 activity was inferred at the 72 kDa band and quantified with ImageJ (Wayne Rasband, NIH, USA). Each zymography experiment was performed in triplicate.
PCa specimens
Soon after the prostate was removed from the patient, we collected a sample of tissue from an area that we believed based on appearance to be compromised by cancer. An adjacent section of this region was sent for histological analysis, and a pathologist expert in urologic malignancies confirmed the presence of neoplasia in 75% of the samples (K.R.L). Frozen prostate-tissue samples were placed in 1.5-mL microtubes, each containing 500 mL of lysis buffer from the mirVana miRNA isolation kit (Ambion, Grand Island, NY) and 5-mm stainless steel beads. The samples were macerated in a TissueLyser LT (Qiagen, Germantown, MD) for 2 min. miRNA was isolated using a mirVana Kit (Applied Biosystems, CA) in accordance with the manufacturer’s instructions, and the nucleic acid concentrations were calculated based on absorbance at 260/280 nM using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, West Palm Beach, FL). For each sample 200 ng of miRNA was reverse transcribed with the High Capacity cDNA Reverse Transcription kit and the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, CA).
Benign tissues obtained through transurethral resection of the prostate in six patients with benign enlargement were employed as controls.
Ethics
Approval for the study was given by the Institutional Board of Ethics (CAPPesq – Comissão de Ética para Análise de Projetos de Pesquisa) under the numbers 054/13 and 352.893/2013.