Materials
Mice
All female BALB/c mice (6–8 weeks, 18–22 g) were supplied by Shanghai Laboratory Animal Center [SLAC, Shanghai, China, Animal Certificate No. SCXK (Hu) 2007-0005]. All the procedures related to animal handling, care and the treatment in this study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
Cell lines
All cell lines used in this study were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). This study used human renal cell carcinoma cells (786-O, OS-RC-2, A-498, Caki-1), a human adrenal gland small cell carcinoma cell line (SW-13) and a human umbilical vein endothelial cell line (HUVEC). Cells were routinely cultured with Dubecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin and streptomycin (Gibco BRC, Paisley, UK).
Human renal cell carcinoma specimens
A total of 23 pairs renal cell carcinoma and normal renal tissue samples were snap-frozen in liquid nitrogen immediately after open radical nephrectomy. The pathologist verified normal and cancer specimens. All protocols were reviewed and approved by the local ethics committee and all patients gave written informed consent.
RNA isolation and reverse transcription PCR
RNA was isolated using Total RNA Isolation reagent (ABgene, Epsom, UK). First strand cDNA was synthesized from 0.5 μg RNA using a reverse transcription kit (Sigma, Poole, Dorset, UK). The quality of cDNA was verified through the amplification and detection of the GAPDH housekeeping gene. VEGI forward and reverse primers (Table I) were designed based on the human VEGI sequence (GeneBank accession no. BD131562). PCR was performed in a GeneAmp PCR system 2400 thermocycler (Perkin-Elmer, Norwalk, CT, USA). Conditions for PCR were 40 sec at 94°C, 60 sec at 55°C, 60 sec at 72°C (35 cycles). PCR products were separated on a 1.4% agarose gel.
IHC staining of human renal and xenograft tumor specimens
Frozen specimens of renal cell carcinoma (n=23), normal renal tissue (n=23) and xenograft tumors (n=24) were cut at a thickness of 6 μm using a cryostat (Leica CM 1900, Leica Microsystems UK Ltd., Buckinghamshire, UK). The nature of the samples was independently verified by two pathologists. After fixation, the sections were blocked with horse serum and probed with or without VEGI antibody (SC-53975) for 1 h. The secondary biotinylated antibody and the avidin-biotin complex were subsequently applied to detect VEGI expression in accordance with the Vectastain Universal Elite ABC kit protocol (Vector Laboratories, Peterborough, UK). After developing color with DAB, the sections were counterstained with Gill’s hematoxylin. Staining was independently assessed by the authors. The monoclonal mouse anti-human-VEGI and anti-human-VEGFR2 antibodies were purchased from LSBio Inc. (LS-C76815 and LS-C92359, respectively, LSBio Inc., Seattle, WA, USA). Monoclonal mouse anti-human-Ki67 and anti-human-CD31 (PECAM) antibodies were purchased from Santa Cruz Biotechnology, Inc. (SC-56320 and SC-133091 respectively, Santa Cruz, CA, USA).
Western blot analysis of VEGI expression
Protein concentration in cell lysates were determined using the DC Protein Assay kit (Bio-Rad) and an ELx800 spectrophotometer (Bio-Tek™). Equal amount of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose sheets. Proteins were then probed with the VEGI antibody (1:1,500) and peroxidase-conjugated secondary antibodies. Monoclonal mouse anti-human-β-actin (1:1,000, SC-130301, Santa Cruz) was used as a housekeeping control. Protein bands were visualized using the Supersignal™ West Dura system (Pierce Biotechnology, Inc., Rockford, IL, USA) and photographed using an UVITech imager (UVITech, g5286Inc., Cambridge, UK).
Construction of VEGI expressing and ribosome transgenes and transfection
The full-length human VEGI coding sequence and hammer-head ribozymes targeting human VEGI was cloned into a mammalian expression plasmid vector (pEF/His TOPO TA, Invitrogen, Inc., Paisley, UK). Full primer sequences are provided in Table I. The recombinant plasmid vectors were transformed into chemically competent TOP10 E. coli (Invitrogen). After verification and amplification, empty control plasmids or plasmids containing VEGI expression sequence or ribozyme transgenes were then transfected into 786-O, SW-13 and SC-OR-2 cells using electroporation (Easyjet, EquiBio Ltd., Kent, UK). After selection with blasticidin, the transfectants were used used for subsequent analysis.
Real-time quantitative polymerase chain reaction (qPCR)
Real-time quantitative PCR was carried out using the iCycleriQ5 system (Bio-Rad, Hemel Hemstead, UK) to determine the level of expression of the VEGI transcripts in the cell lines. GAPDH was used as a housekeeping control.
Cell growth assay
This was based on a method we previously described (33). Briefly, cells were plated into a 96-well plate (2,500 cells/well). Cell growth was assessed after 1, 3 and 5 days. Crystal violet was used to stain cells and absorbance was determined at a wavelength of 540 nm using a spectrophotometer (Bio-Tek, Elx800, UK).
Flow cytometric analysis of apoptosis
All cells including those floating in the culture medium were harvested after a period of incubation. The apoptotic population of the cells was determined using Vybrant® Apoptosis Assay kit (Invitrogen) and flow cytometry (CyFlow® SL, Partec GmbH, Münster, Germany).
Wounding assay
The migration of cells in a monolayer across a wounded surface of a near-confluent cell monolayer was examined. Cells at a density of 50,000/well were seeded into a chamber slide and allowed to reach near confluence. The monolayer of cells was then scraped with a fine gauge needle to create a wound of ∼200 μm. The movement of cells to close the wound was recorded using a time lapse video recorder and analysed using the motion analysis feature of the Image J software (NIH, USA).
Motility assay using Cytodex-2 beads
Cells (1×106) were incubated with 100 μl of cytocarrier beads overnight. The beads were washed twice to remove dead cells and then resuspended. Beads/cells (100 μl) were transferred into each well of a 24-well plate. After incubation for 4 h, the cells were fixed in 4% formalin and stained with 0.5% crystal violet before counting.
Invasion assay
This was modified from a method we prevously described (33). Transwell inserts with 8-μm pore size were coated with 50 μg Matrigel (BD Matrigel™ basement membrane matrix) and air-dried. After rehydration, 20,000 cells were added to each well. After 96 h cells that had migrated through the matrix to the other side of the insert were fixed, stained and then counted under a microscope.
Cell-matrix adhesion assay
Cell-matrix adhesion was assessed in a standard manner. Cells (40,000) were added in each well of 96-well plate, previously coated with Matrigel (5 μg/well). After 40 min of incubation, non-adherent cells were washed off using BSS buffer. The remaining adherent cells were then fixed and stained with crystal violet. The number of adherent cells was then counted.
Endothelial cell differentiation assay: tube formation on Matrigel (<xref rid="b34-ijo-42-05-1664" ref-type="bibr">34</xref>)
This was based on a Matrigel-sandwich tubule forming assay developed in our laboratory. Briefly, 200 μg of cold Matrigel solution dispersed in 100 μl (Becton-Dickinson, Bristol, UK) was added to a 96-well plate and allowed to air-dry at 37°C. Following rehydration of Matrigel, it formed a thin bottom layer. HUVEC cells (2.5×104) per well were seeded onto a 96-well plate and allowed to attach to the bottom of the well (4–6 h). After that, 200 μg of cold Matrigel solution dispersed in 100 μl was loaded to each well again, followed by incubation at 37°C for 3 h when the second layer of Matrigel solidified. Finally, 10,000 tested cells (different renal cell carcinoma cell lines) were added and incubated at 37°C in a humidified chamber with 5% CO2. Following incubation for 7 or 20 h, cultures were observed and photographed (Nikon, Tokyo, Japan).
Tumor xenografts in Nu/Nu nude mice
Twenty-four mice were divided into three groups (786-Owt Group, 786-OpEF/His Group and 786-OVEGIexp4 Group). Each mouse was inoculated subcutaneously at the right flank with 786-O cells (5×106) in 0.1 ml of PBS for tumor development. The mice were kept in individually ventilated cage (IVC) systems at constant temperature and humidity with 4 animals in each cage. The date of tumor cell inoculation was denoted as day 0. The data of tumor growth and normal behavior such as mobility, visual estimation of food and water consumption, body weight gain/ loss (body weights were measured twice weekly), eye/hair matting and any other abnormal effect were collected every three days. Tumor sizes were measured each three days in two dimensions using a caliper and the volume was expressed in mm3 using the formula: V = 0.5 a x b2 where a and b were the long and short diameters of the tumor, respectively.
Statistical analysis and software
The mean optical density (MOD) feature of RT-PCR and western blot analysis was analysed using software package Image-Pro Plus 6.0 (Media Cybernetics, USA). The method was as follows: every image prepared for comparison was changed to grayscale image. Then, selecting ten points in the comparison region randomly, the MOD of the point was analyzed using the software. The length of cell migration and HUVEC was measured by software Image J (NIH). The distance of HUVEC cells was calculated along the long axes in each compared image.
All statistical analysis was performed using the SPSS 16.0 software. Two sample t-tests was used for normally distributed data. Fisher’s exact test was used for analyzing immunohistochemical staining in renal tissues. Differences were considered to be statistically significant at P<0.05.