Primary cell cultures and identification
The samples used in this study were obtained after obtaining informed consent of each patient and with the approval of the Sun Yet-sen University Ethics Committee. Briefly, ameloblastoma tissues were minced and incubated overnight in Dulbecco's modified Eagle medium (DMEM, Invitrogen, CA, USA) containing 1 mg/mL collagenase I (Invitrogen) at 37°C. Collagenase-digested tissues were plated onto 35 mm dishes coated with collagen I (Invitrogen) in DMEM containing 10% fetal calf serum, 200 μg/ml streptomycin, and 200 IU/ml penicillin, and incubated at 37°C with 5% CO2. When the cells were confluent, they were divided again and used for the ensuing experiments.
Immunocytochemistry was used to confirm the epithelial origin of the ameloblastoma cells using the SP method as described by the manufacturer (Maixin, Fuzhou, China). The primary antibodies were anti-cytokeratin 14, 16, and 18 and anti-vimentin (Maixin). Immunofluorescence was used to detect the expression of MMP-2, as described by the product's manufacturer.
Plasmid construction and transient transfection
To generate the plasmid vector, pRNA-MMP-2, pRNA-U6.1/neovector (Genscript, NJ, USA) containing a cGFP sequence was used. MMP-2 shRNA contains a complement of a 21-nucleotide sequence (tgtgctgaaggacacactaaa, GenBank NM-004530), which was separated by a 7-nucleotide non-complementary spacer (CCACACC). A control vector (pRNA-neg) was constructed in the same way using a 21-nucleotide sequence (gattcaggtgtagaacgagca). These sequences were confirmed using nucleotide BLAST to ensure that there was no homology with any other known human gene. These annealed sequences were inserted into the pRNA-U6.1/neo backbone after digestion with BamH1 and HindIII. After amplification, all vector constructs were verified by sequencing.
The pcDNA3 vector (Genscript) containing an enhanced green fluorescence protein (EGFP) sequence was employed to generate the plasmid vector, pcDNA-TIMP-2. The cDNA encoding TIMP-2 was obtained via RT-PCR. The primer sequence for TIMP-2 (723 bp, GenBank NM003522) containing EcoRI and XhoI was as follows: forward 5'cgatgaattcatgggcgccgcggcccgc3'; reverse 5'cgatctcgagttattatgggtcctcgatgagaaac3'. TIMP-2 cDNA was subcloned into the clone site of the pcDNA3. Following amplification, vector constructs were verified by sequencing.
The constructed plasmids were transiently transfected into the cultured ameloblastoma cells using Lipofectamine Plus reagent (Invitrogen), according to the manufacturer's instructions. Transfected cells were subsequently used in the following experiment.
Detection of MMP-2/TIMP-2 activity
MMP-2/TIMP-2 activity in the culture medium of the ameloblastoma cells was detected by zymography according to the method reported by Kleiner et al.
]. Briefly, 10 μl of the medium from serum-free ameloblastoma cell cultures was mixed with the same volume of sample buffer and applied to a 10% (wt/vol) polyacrylamide gel containing 1 mg/ml gelatin. Gels were incubated in 2.5% Triton X-100 for 45 min after electrophoresis, then incubated at 37°C overnight in a digestion buffer. Gels were stained and destained. The bands were analyzed by an auto-imaging analysis system (Kontron IBAS2.0, Germany). All densitometry measurements were made between samples in the same gel to ensure comparability.
For detection of TIMP-2 activity, all procedures were similar to the detection of MMP-2 activity (described above), except that the gel contained 1% (wt/vol) MMP-2 (Sigma, St. Louis, MO, USA).
RNA preparation and RT-PCR
Total RNA was extracted using an RNeasy mini kit (Qiagen), according to the manufacturer's instructions. RT-PCR was performed using one-step RT-PCR assays (Qiagen). Specific primers for detecting mRNA transcripts of the MMP-2 or TIMP-2 gene were as follows: MMP-2 (NM004530), 5'-AGCCACCCCTAAAGAGATCC-3' and 3'GTTCTAAGGCAGCCAGCAGT-5'; TIMP-2 (NM003255), 5'-ATTTGACCCAGAGTGGAACG-3' and 3'-TCCTTCGGCGAGTTTATGGA-5'; and GAPDH (NM008084), 5' GGTCGGAGTCAACGGATTTGGTCG-3' and 3'-CCTCCGACGCCTGCTTCACCAC-5'.
Transcript levels were normalized according to GAPDH transcripts and the products were resolved by agarose electrophoresis. The intensity was quantified by image-analysis computer software (NIH Image).
Western blotting was performed to detect MMP-2 and TIMP-2 proteins (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were harvested by trypsinization and lysed with a radio-immune precipitation assay (RIPA) lysis buffer (Santa Cruz Biotechnology). Total protein concentrations were measured by the Bradford method (Bio-Rad). Aliquots (30–50 μg) of cellular proteins were resolved by SDS-PAGE (10%), then electrotransferred onto PVDF membranes and immunoprobed. The protein-antibody complexes were detected by chemiluminescence (CSPD; Tropix, Bedford, MA, USA), according to the manufacturer's protocol (Applied Biosystems, MA, USA). The GAPDH gene was used as an internal control and the band intensity was quantified.
In vitro cell invasion
The invasive ability of ameloblastoma cells was assayed in transwell cell chambers (Costa, Cambrige, MA, USA), according to the method reported by Kido et al.
]. Briefly, polycarbonate filters with an 8.0 μm pore size were precoated with fibronectin on the lower surface. Matrigel was applied to the upper surface of the filters (5 μg/filter). Ameloblastoma cell suspensions (100 μl with 2 × 106
cells/ml) that had or had not been transfected were added to the upper compartment and incubated for 72 h at 37°C at 5% CO2
. The filters were fixed with methanol and stained with Giemsa stain. The cells invading the lower surface through the Matrigel were manually counted under a microscope. The rate of invasion was calculated by the following equation: (number of cells invading the lower surface in the control group – number of cells invading the lower surface in the treated group)/number of cells invading the lower surface in the control group × 100%.
All experiments were performed in triplicate. Data are expressed as the mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to compare differences between treatment and control cells. A p < 0.05 was considered significant.