Reduced Expression of Metastasis Suppressor-1 (MTSS1) Accelerates Progression of Human Bladder Uroepithelium Cell Carcinoma

Materials and Methods

BUCC specimens. A total of 68 (41 males and 27 females) pairs of BUCC and normal bladder tissue samples from newly diagnosed patients who underwent total cystectomy or transurethral resection of bladder cancer from May 2014 to December 2015 at Peking University Cancer Hospital were snap-frozen in liquid nitrogen immediately after surgery. The average age of patients was 52.7±10.4 (range=26-78) years. In the cohort, 52 (76.5%) and 16 (23.5%) cases were staged as non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC), respectively. Pathological grades of the cohort were low grade in 48 cases (70.6%) and high grade in 20 (29.4%). The present study was performed according to the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Cancer Hospital, and written informed consent was obtained from each patient.

Cell cultures. Bladder cancer cell line 5637 was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured routinely in RPMI-1640, supplemented with 5% fetal bovine serum, 100 U/ml penicillin and 50 μg/ml streptomycin (all Gibco, Carlsbad, CA, USA). All cell lines were grown in a humidified incubator at 37°C and 5% CO₂.

Expression vectors and lentiviral infection. Lentiviral transfected with MTSS1 expression plasmid (NM_014751) (GV287-RBM5) and plasmids containing scrambled control sequence (GV287) were constructed by Genechem Company (Genechem, Shanghai, China). Bladder cancer 5637 cells were infected with MTSS1 overexpression lentiviral vectors and negative control lentiviral vectors according to the manufacturer's instruction, with 1 μg DNA for 4×10⁵ 5637 cells in a 24-well plate. Five hours after the transfection, the cell culture medium was replaced with 10% fetal bovine serum.

Bromodeoxyuridine labeling. For the bromodeoxyuridine (BrdU) labeling, cells were plated into 96-well culture plates at a 2×10⁴ cells per well in 100 μl cell culture medium and maintained at 37°C. The cell growth rate was determined using a BrdU cell proliferation ELISA kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions after 1 and 4 days. Each experiment was performed in triplicate and repeated three times independently. Cell-cycle analysis. For cell-cycle analysis, cells were seeded on 6-well plates at 80% confluence and incubated at 37°C for 24 h. The cells were harvested, washed with ice-cold phosphate buffered saline and resuspended in ice-cold 70% ethanol for 30 min. The cells were then treated with 10 mg/ml RNase at 37°C for 30 min. The cells were stained with 10 mg/ml propidium iodide (Sigma, St. Louis, MO, USA) for 30 min. The DNA content was measured by flow cytometry.

Colony formation in soft agarose gel. For the cell colony formation assay, cells were seeded into 6-well plates at 800 cells per well and cultured at 37°C for 14 days. The medium was replaced every 3-4 days. Cells were washed twice with phosphate-buffered saline, fixed with paraformaldehyde, stained with Giemsa (Sigma) for 20 minutes, and washed with ddH₂O three times. The plates were photographed with a microscope. Each experiment was performed in triplicate and repeated three times independently.

Cell apoptosis detection. For cell apoptosis detection, cells were seeded on 6-well plates at 80% confluence and incubated at 37°C for 24 h. The cells were harvested, washed with ice-cold phosphate-buffered saline and resuspended in staining buffer. Flow cytometry was performed to analyze apoptosis. An annexin V-allophycocyanin (APC) stain assay (eBioscience, Vienna, Austria) was performed using the manufacturer's protocol, and the apoptotic cells were analyzed by flow cytometry.

RNA extraction and quantitative real-time polymerase chain reaction. Total RNA was extracted from tissues or cells using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). FastQuant RT Kit (With gDNase kit and SuperReal PreMix Plus (SYBR Green) (TIANGEN BIOTECH, Beijing, P.R. China) were used. The PCR primers used were as follows: MTSS1 forward: 5’-ACTGGGAATCTGACCACTAT-3’; MTSS1 reverse: 5’-GACCTGACTGCTAAGGGAC-3’; glyceraldheyde 3-phosphate dehydrogenase (GAPDH) forward: 5’-TGACTTCAACAGCGACACCCA-3’, GAPDH reverse: 5’-CACCCTGTTGCTGTAGCCAAA-3’. PCR cycling conditions were as follows: an Initial denaturation period at 95°C for 15 min, followed by a two-step PCR program consisting of denaturation at 95°C for 10 s and annealing/extension at 60°C for 32 s for 40 cycles. All samples were normalized against the internal control (GAPDH) and analyzed using the 2^−ΔΔCt method.

Western blot analysis. Total protein was extracted from the cells or tissue using lysis buffer containing protease inhibitors. Thirty micrograms of protein from each sample were loaded on 10% sodium dodecyl sulfonate polyacrylamide gel electrophoresis. The gels were electrophoresed, and then transferred onto polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK), and blocked with 10% non-fat milk in Tween/Tris-buffered salt solution [20 mM Tris–Cl, pH 7.5, 0.15 M NaCl and 0.05% Tween-20] for 1 h. Following incubation with the antibody to MTSS1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight, the membrane was washed and incubated with IRDye® 800CW Goat anti-rabbit IgG secondary antibody at room temperature for 2 h followed by detection using a LiCor Odyssey gel imaging scanner (LI-COR Biosciences, Lincoln, NE, USA). To verify equal loading of protein, the blots were re-probed with a primary monoclonal antibody against GAPDH (Proteintech Group, Inc., Chicago, IL, USA).

Statistical analysis. Quantitative analysis of western blot was performed using the GelDoc-2000 Imaging System (UVi Company, Cambridge, UK). For protein expression level, the protein ratio (band density of study protein/band density of GAPDH) was considered as 100% in normal bladder tissues, and that of the other tissues was expressed as a percentage of that of the normal bladder tissues. Comparison between groups was performed using one-way analysis of variance followed by all pairwise multiple comparison procedures using Bonferroni test and Student t-test or nonparametric test. In addition, Spearman correlation was used for comparison and estimation of correlations between MTSS1 protein expression levels and clinicopathological parameters. Survival curves were constructed using the Kaplan–Meier method. All data are presented as the mean±standard deviation, and a value of p<0.05 was regarded as significant.

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Figure 1.

Lower expression of metastasis suppressor 1 (MTSS1) mRNA in patients with bladder uroepithelium cell carcinoma (BUCC). The relative expression of MTSS1 mRNA in BUCC tissues was significantly lower than that of normal bladder tissues. Furthermore, the mRNA level of MTSS1 was higher in non-muscle-invasive bladder cancer (NMIBC) than muscle-invasive bladder cancer (MIBC), *p<0.05 vs. Normal, ^#p<0.05 vs. NMIBC (A). Furthermore, the mRNA level of MTSS1 was higher in low pathological grade cancer than high pathological grade cancer tissues, *p<0.05 vs. Normal, ^#p<0.05 vs. low grade. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acted as an internal loading control. Each sample was analyzed in triplicates.