The study included a total of 30 patients receiving first line platinum containing combination chemotherapy for locally advanced (T4B, N2-3) and/or metastatic (M1) transitional cell carcinoma (TCC) at the Department of Oncology, Aarhus University Hospital, Denmark, in the 10-year period between 1991 and 2001. All patients had histologically confirmed transitional cell carcinoma of the urothelial tract (urethra, bladder, ureters or renal pelvis). All patients received cisplatin-based combination chemotherapy schedules (Additional file 1: Tables S1 and Additional file 2: Table S2) and were included in either one of four phase II protocols, one phase III protocol, or were treated with the standard regimen at the relevant time (MVAC or GC). Patients were treated to a maximum of 6 cycles unless progression or unacceptable toxicity appeared. Patients receiving standard treatment were prospectively followed under the same conditions as patients in protocols . Patients presenting with locally advanced disease, obtaining a significant partial or complete response to chemotherapy, were offered consolidating surgery or, more often, radiotherapy when applicable .
Bladder cell lines UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197, 575A  (provided by Professor Dan Theodorescu, University of Colorado Comprehensive Cancer Center) were re-authenticated via STR analysis using the Cell-ID-system (G9500, Promega, Nacka, Sweden), products were analyzed using an Applied-Biosystems 3130 Genetic Analyzer. No Mycoplasma contamination was detected using nested PCR-based Mycoplasma detection. SLT4, 253JBV, RT4, CLR2169 cells were cultured in DMEM + with L-glutamine (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco) and 100 U/mL streptomycin (Gibco). UMUC9, UMUC14, 253JBV, HT1197, 575A cells were cultured in MEM with L-glutamine (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 1 mM Sodium Pyruvate + 0,1 mM nonessential amino, 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco) and 100 U/mL streptomycin (Gibco). All cells were maintained in a humidified atmosphere at 37°C and 5% CO2.
miRNA molecules (Ambion, Austin, TX, USA) and LNA knockdown molecules (Exiqon) were reverse Transfected using Lipofectamine 2000 (Invitrogen) according to manufacturer's guidelines. Non-targeting miRNA (Ambion, Austin, TX, USA) and non-targeting LNA molecules (Exiqon, DK) were used as controls. In short 4x103 cells in 100 μL culture medium were added to wells of 96 well plates containing 50 μL miRNAs or LNA antagonism giving a final concentration of 20 nM and 40 nM respectively and then incubated at 37°C and 5% CO2. Cisplatin (Sigma Aldrich, Germany) was added 48 h post transfection in experiments studying the effect of chemotherapy, by first removing 50μL from each well and then adding 100μL new medium containing cisplatin at 2 times the desired concentration and then incubated for an additional 48 h at 37°C and 5% CO2. Finally viability was measured using the MTT assay (see section: Cell viability assay). mercury LNA™ microRNA inhibitors (EXIQON) used: hsa-miR-27a (410167-00), hsa-miR-138 (412560-00), hsa-miR-296-5p (410171-00), hsa-miR-642 (410409-00), hsa-miR-886-3p(410445-00) and mercury LNA™ microRNA inhibitors negative control A (199004-04). The miRNA molecules used for transfection are chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNA molecules: hsa-miR-27a (PM10939), hsa-miR-138 (PM11727), hsa-miR-296-5p (PM10609), hsa-miR-410 (PM11119), hsa-miR-642 (PM11477), hsa-miR-886-3p(410445-00), Pre-miR™ miRNA Precursor Molecules Negative Control #1(AM17110). Prior to miRNA transfection experiments we performed transfection with a cy3 labeled siRNA molecule to monitor successful uptake under fluorescence microscope.
Isolation of RNA
Total RNA was isolated from formalin-fixed paraffin embedded (FFPE) tissues specimens as follows. Haematoxylin- and eosin-stained (HE) sections of the FFPE blocks were examined and areas with carcinoma cells circled and the section was photocopied onto transparent paper. Using this as a guide, an appropriate area of the block was sampled using a disposable punch biopsy needle (1.5 mm, Miltex GmbH, Germany). The sample cylinder was dispensed and minced into small pieces with a scalpel. Total RNA (range 1200 ng – 2300 ng) was then prepared using the miRNeasy® FFPE Kit (Qiagen, Germany), essentially as specified by the manufacture. The miRNeasy FFPE kit is specifically designed for FFPE material and contains reagents to reverse formalin cross linking of RNA. Furthermore the kit is developed to allow purification of all usable RNA larger than 18 nucleotides.
Cell line RNA was isolated from 6-well and 24-well plates using RNeasy spin columns (Qiagen, Hilden, Germany), as recommended by the manufacturer. All RNA samples were quantified using an Infinite® 200 PRO NanoQuant spectrophotometer (Tecan, Switzerland).
miRNA profiling using Taqman Micro fluidic cards
Quantification of miRNAs from tissue samples was carried out by a two-step procedure using the Taqman LDA Human microRNA Panel v3.0 (Micro fluidic card, Applied Biosystems, Foster City, CA) according to the manufacturer's protocols. Taqman LDA Human microRNA Panel v3.0 was designed using Sanger miRBase v14. Briefly, 500 ng of total RNA was used for multiplexed RT reactions followed by pre-amplification. Cycling conditions were chosen according to the manufacturer's protocols. The ABI7900 HT platform with TLDA upgrade was used and data was analyzed using RQ Manager Software provided by Applied Biosystems.
Quantitative real-time PCR
Quantification of miRNAs by Taqman real-time PCR was carried out as described by the manufacturer (Applied Biosystems). Briefly, 500 ng of template RNA was reverse transcribed by using the Taqman MicroRNA Reverse Transcription Kit and the multiplex RT primer pools containing miRNA-specific stem-loop primers (Applied Biosystems). Diluted RT product (1.5 μL) was introduced into the 20 μL PCR amplifications, which were incubated in 384-well plates on the ABI 7900HT thermo cycler (Applied Biosystems) at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Gene expression was normalized using expression of miRNA-193b. Taqman® MicroRNA Assays (Applied Biosystems, Foster City, CA) used: hsa-miR-27a (Assay ID000408), hsa-miR-138 (Assay ID002284), hsa-miR-193a-5p (Assay ID 002281), hsa-miR-193b (ID 002367), hsa-miR-296-5p (Assay ID 000527), hsa-miR-410 (Assay ID001274), hsa-miR-492 (Assay ID001039), hsa-miR-639 (Assay ID001583), hsa-miR-642 (Assay ID 001592), hsa-miR-886-3p(Assay ID002194), hsa-miR-944 (Assay ID002189).
Cell viability assay
The viability of sub-confluent cells was analyzed by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The assay was performed in 96 well plates with 200μL/well. 100 μL culture medium was carefully removed and 25 μL MTT solution was added (1 g MTT (Sigma M5655) dissolved in 200 mL D-PBS.) and stored shielded from light 1.5 h at 37°C and then 100μL solubilization (50% dimethylformamide, 20% SDS) buffer was added and left protected from light overnight. Readout was done using a micro plate reader (Lab systems Multiscan MCC/340) at 540 nm. Absorbance at 692 nm was used as reference.
All patients gave their written informed consent, and the study was approved by the Central Denmark Region Committees on Biomedical Research Ethics (1994/2920).
Selection of differentially expressed miRNAs were based on t-test statistics (p < 0.05) and simultaneously on assessment of false discovery rates (FDR) to correct for multiple testing. We used Significance Analysis of Microarrays (SAM) software for estimating FDR. In the analysis of treatment response we used a maximum of 10% in FDR as cutoff and in the analysis of survival we used a maximum of 50% in FDR as cutoff. FDRs for the individual miRNAs are reported.