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).
Cell Lines and Cell Culture
Human renal cancer cell lines A-498, ACHN, Caki-1, Caki-2, and a normal renal cell line (HK-2) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Integrity of the cell lines was confirmed by the ATCC using 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), 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 lines A-498 and ACHN were 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.
Genistein Treatment of A-498 Cells
A-498 cells (60–70% confluent) were treated with genistein (25 µM and 50 µM ). Genistein (Sigma-Aldrich Corp., St Louis, MO, USA) was dissolved in DMSO, and cells treated with vehicle (DMSO) served as control. Fresh genistein was administered everyday with a change of medium, and the cells incubated for 4 days.
Quantitative Real-time PCR
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 3s and 60°C for 30s according to the TaqMan Fast Universal PCR protocol. Total microRNA was extracted using a miRNeasy kit from Qiagen (Valencia, CA, USA). For miRNA real-time experiments the cDNA strand was synthesized using an Applied Biosystems Taqman MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA), with 200 ng of total extracted miRNA. RNU48 was used as an endogenous control. It was also used as an endogenous control for real-time PCR experiments, where miRNAs were isolated from formalin-fixed paraffin-embedded (FFPE) renal samples. Total miRNA was extracted from FFPE samples using a miRNA FFPE kit from Qiagen.
Micro Dissection of Renal Cancer Tissues and RNA Extraction from FFPE Human Renal Tumor Samples
Adjacent normal and cancerous renal tissues were obtained from 29 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 micro dissection 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-23b-3p were assessed by the Taqman miR assay as described above. Following PCR, relative miR-23b-3p expression levels in cancerous regions were normalised to their adjacent normal counterparts.
A498 and Caki-2 cells were transiently transfected with either miRNA-23b-3p inhibitor (mirVana® miRNA inhibitor Applied Biosystems, Assay ID no. MH10711) or anti-miR negative control #1 (from Ambion, Austin, TX, USA Catalog no. AM17010), 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 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, 100x, 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(7-AAD) 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 proteins detected by chemiluminescence. Antibodies, including PTEN (cat. no. 9552S) and Akt (cat. no. 4691S) were purchased from Cell Signaling (Danvers, MA, USA), PI3-kinase (cat no. ab69870) and IL-32 (cat no. ab62580) were from Abcam (Cambridge, MA, USA), whereas GAPDH (cat. no. sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
The protein expression levels were quantified by optical densitometry using ImageJ Software version 1.36b (http://rsb.info.nih.gov/ij/
). Fold change was calculated as the ratio between the net intensity of each sample transfected with anti-miR-23b-3p divided by GAPDH and the negative control transfected samples divided by the GAPDH (Anti-miR-23b-3p transfected samples/GAPDH)/(Neg Control miR transfected samples/GAPDH).
Luciferase Reporter Assay
Cells in 24-well plates were transfected with 30 nM pre-miR negative control (NC) or pre-miR-23b-3p (Applied Biosystems) and PTEN construct (Catalog no. HmiT015535, Genecopoeia, Rockville, MD, USA) or control construct, pEZX-MT01 (Genecopoeia) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The control construct lacked miR-23b-3p target sites. All transfection experiments were performed in triplicate. Luciferase activity was assayed at 48 h after transfection, using a dual-luciferase reporter assay system (Promega).
Immunostaining was done on renal cancer tissue slides [made from formalin-fixed, paraffin-embedded (FFPE) renal cell cancer tissue blocks using a microtome (Leica)]. The slides were deparaffinized and antigen retrieval was carried out by microwaving the slides in 10 mmol/L sodium citrate buffer. Slides were incubated overnight with anti-PTEN antibody (Cell signalling). The staining was done using the LABVISION Corporation (Thermo Fisher Scientific) Anti-Rabbit Staining System (LOT-RHD 80924) as per manufacturer’s instructions.
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. Chi-square test was performed to determine the correlation between miR-23b-3p expression levels and PTEN protein expression levels in tissue samples.