Formalin-fixed, paraffin-embedded (FFPE) renal 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 lines and cell culture
Human renal cancer cell lines A-498, Caki-1, Caki-2, and normal renal cell line HK-2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines were checked by ATCC through DNA (STR) profiling. Normal renal HK-2 cells were cultured as a monolayer in Keratinocyte Serum Free Medium (K-SFM) supplemented with 0.05 mg/ml bovine pituitary extract (BPE), 5 ng/ml human recombinant epidermal growth factor (EGF) (Life Technologies/Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA), 50 mg/ml penicillin and 50 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). The renal cancer cell line A-498 was cultured as a monolayer in Eagle's Minimum Essential Medium, (UCSF Cell Culture Facility, San Francisco, CA, USA). Caki-1 and Caki-2 were cultured the same way in McCoy's5A media (UCSF Cell Culture Facility, San Francisco, CA, USA). All cell lines were maintained in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37°C. Subconfluent A-498cells (60–70% confluent) were treated with Genistein (25 µM). Genistein (Sigma-Aldrich Corp., St Louis, MO, USA) was dissolved in DMSO, and cells treated only with vehicle (DMSO) served as control. Fresh genistein was administered everyday along with a change of medium, and the cells incubated for 4 days.
Quantitative real-time PCR
First-strand cDNA was prepared from total RNA (1 µg) using a reverse transcription system (Promega, Madison, WI, USA). Total RNA was extracted using an RNeasy mini kit from Qiagen (Valencia, CA, USA). For real-time polymerase chain reaction (PCR), complementary DNA was amplified with Inventoried Gene Assay Products containing two gene-specific primers and one TaqMan MGB probe (6-FAM dye-labeled) using a TaqMan Universal Fast PCR Master Mix in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Thermal cycling conditions included 95°C for 20 seconds(s), 40 cycles of 95°C for 3 s and 60°C for 30 s according to the TaqMan Fast Universal PCR protocol. GAPDH was used as an endogenous control. For miRNA real-time experiments the cDNA strand was synthesised using Applied Biosystems Taqman MicroRNA Reverse Transcription kit, with 200 ng of total extracted miRNA. RNU48 was used as an endogenous control. For real-time PCR experiments, where miRNAs were isolated from formalin-fixed paraffin-embedded (FFPE) renal samples, let7-f was used as an endogenous control. Total miRNA was extracted from FFPE samples using a miRNA FFPE kit from Qiagen.
PCR arrays through real-time PCR
To study the expression profile of genes involved in various cellular pathways, pathway-specific PCR arrays (cell cycle array, apoptosis array, MAP kinase array, SABioscience) were used according to the manufacturer's instructions. Data was analyzed with the manufacturer's software.
Micro dissection of renal cancer tissues and RNA extraction from FFPE human renal tumor samples
Adjacent normal and cancerous renal tissues were obtained from 39 representative FFPE tissue blocks of radical nephrectomy specimens from the Pathology Department of the Veterans Affairs (VA) Medical Center of San Francisco. The blocks were from kidney cancer patients who were operated on at the VA Medical Center between 1980–2009. Sections (4 µm) of the blocks were prepared, H&E stained, and slides were reviewed by a board certified pathologist to mark the normal and cancer areas. Subsequently, 12 µm slides were made from the blocks and microdissection was performed using the marked H&E stained slides as a template. MicroRNA extraction was done using a Qiagen FFPE miRNA extraction kit. The levels of miR-21 were assessed by the Taqman miR assay as described above. Following PCR, relative miR-21 expression levels in cancerous regions were normalised to their adjacent normal counterparts.
A498 and Caki-2 cells were transiently transfected with either an inhibitor of miRNA-21 or anti-miR negative control #1 (from Ambion, Austin, TX, USA), using X-tremeGENE siRNA transfection reagent (Roche Diagnostics Indianapolis, IN, USA) according to the manufacturer's protocol. In brief, cells were seeded in 6 well plates (Nunc, Roskilde, Denmark) 24 h before transfection and were transiently transfected at a confluency of 40–50%. Mock transfection, with the transfection reagent, was also used as a control. The transfection mixture was dissolved in Opti-MEM serum-free media (UCSF Cell Culture Facility, San Francisco, CA, USA) and at the time of transfection cells were seeded in Eagle's Minimum Essential Medium (UCSF Cell culture facility), with 10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA) and no antibiotics. On the following day the media was changed to Eagle's Minimum Essential Medium containing both FBS and 1% antibiotic (penicillin–streptomycin, 100×, UCSF Cell Culture Facility). Cells were pelleted after 72 h of transfection for flow cytometry, RNA and protein extraction.
Cell cycle analysis
Cell cycle analysis was performed 72 h after transfection. The cells were harvested, washed with cold PBS, UCSF Cell Culture Facility, and resuspended in the nuclear stain 4′,6-diamidino-2-phenylindole (Beckman Coulter, Brea, CA, USA). Stained cells were immediately analysed with a flow cytometer (Cell Lab Quanta SC; Beckman Coulter).
For apoptosis, cells at 72 h after transfection were dual stained with the viability dye 7-amino-actinomycin D and Annexin V-FITC using an Annexin V-FITC/7-amino-actinomycin D kit (Beckman Coulter) according to the manufacturer's protocol. Stained cells were immediately analysed by flow cytometry (Cell Lab Quanta SC; Beckman Coulter).
Migration and invasion assays
A cytoselect 24-well cell migration and invasion assay kit (Cell Biolabs, Inc., San Diego, CA) was used for migration and invasion assays at 72 hrs after transfection (8 µm, Colorimetric format) according to the manufacturer's protocol.
Whole-cell extracts were prepared in radioimmunoprecipitation assay buffer (RIPA; Thermo Scientific, Rockford, IL, USA; 50 mmol l–1 Tris (pH 8.0), 150 mmol l–1 NaCl, 0.5% deoxycholate, 0.1% SDS and 1.0% NP-40) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein assays were performed using a BCA Protein assay kit (Pierce/Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. Total protein (40 µg) was electrophoresed in 12% SDS–PAGE gels, and Western blotting was carried out using standard protocols and detected by chemiluminescence. All antibodies, which included p21(cat. no. 2552), p38 MAPkinase (cat. no. 9212) and cyclinE2 (cat. no. 4132) were purchased from Cell Signaling (Danvers, MA, USA), whereas GAPDH (cat. no. sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA).
Genistein treatment of A-498 cells and sub-cutaneous injection in nude mice
As mentioned above, A-498 cells were treated with 25 µM genistein for 96 hours. DMSO (vehicle) treated cells served as the control. After 96 hours the cells were pelleted and ~2 million cells were injected subcutaneously (100 µl) in the right flank of nude mice (4- to 5-week old; Charles River Laboratories). Two sets of mice, with three mice each, were used for this experiment. One set represented the vehicle (DMSO) controls and the other genistein treated cells. Tumors from control and genistein treated cells were excised after 8 weeks when the DMSO treated control mice developed tumors approximately 800 mm3 in volume (length-16 mm, breadth-10 mm). Total RNA was extracted by homogenizing the tumors in Qiazol and extracting RNA using the manufacturer's protocol (Qiagen). Expression of miR-21 was measured by real-time PCR.
Statistical analysis was performed using StatView version 5.0 for Windows. Student's t-test was used to compare the different groups. p-values of <0.05 were regarded as statistically significant.