Reagents and antibodies
Streptavidin-perosidase (SP) and diaminobenzidine (DAB) kits were purchased from Maixin Biotechnology Company (Fuzhou, China). The following primary antibodies were used for Western blot analysis: anti-CD36 (Santa Cruz, CA, USA), anti-E-cadherin, anti-TGF-β, anti-vimentin, anti-snail, anti-slug, anti-twist (Proteintech, IL, USA), and anti-GAPDH (Goodhere Biotechnology, Hangzhou, China).
Human cervical cancer tissues and cell culture
We acquired 133 cases of cervical cancer (CC) and 47 cases of normal cervical tissues between January 2011 to December 2016 from Department of Pathology of the Eighth Affiliated Hospital, Sun Yat-sen University and the Affiliated Hospital of Guangdong Medical University. The diagnoses were conducted by three professional pathologists, and the study was approved by the Institutional Research Ethics Board of the Eighth Affiliated Hospital, Sun Yat-sen University. We purchased the human cervical cancer cell lines C33a, Hce1, HeLa, and SiHa from the China Center for Type Collection (CCTCC) (Wuhan, China). Cell lines were cultured in DMEM (Gibco, CA, USA) medium containing 10% fetal bovine serum (FBS, Sera Gld, Amarica), 100 U/mL of penicillin, and 100 U/mL of streptomycin. All of the cells were incubated in a humidified incubator in 5% CO2 in compressed air at 37 °C.
Immunohistochemistry
Paraffin blocks were cut into 4-μm sections and treated routinely following the reagent instructions. After microwaving in citrate buffer for 5 min, the slides were incubated with anti-CD36 at room temperature. The sections were then incubated with a secondary antibody (MaximBio Company, Fuzhou, China), labeling was monitored using diaminobenzidine (Maxim-Bio Company, Fuzhou, China), and hematoxylin was used to stain the sections. We scored expression in accordance with the intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining), and the percentage of cervical cancer cells that were stained (0, none stained; 1, < 10% stained; 2, 10–50% stained; 3, > 50% stained; 4, > 75% of all of the cervical cancer cells stained). If the product of multiplying staining intensity by the percentage of positively stained cervical cancer cells was ≥ 2, it was regarded as positive (+).
Transfection with small interfering RNA (siRNA)
Homo sapiens CD36 siRNA was obtained from Guangzhou RiboBio Biological Technology (Guangzhou, China), and it targeted the sequence 5′-ACGTATAAGGACCTCTTTG-3′. HeLa and SiHa cells were seeded at 2 × 105 cells/well in six-well plates. We transfected HeLa and SiHa cells with CD36 siRNA or control siRNA (sense strand, 5′- UUCUCCGAACGUGUCACGU TT-3′; antisense, 5′-ACGUGACACG UUCGGAGAATT-3′) with Lipo3000 at a final concentration of 100 nM, and incubated the cells at room temperature for 15 min. The complex was then added to the culture medium for subsequent experiments.
Plasmid construction and transfection
pIRES2-ZsGreen1-CD36 was constructed and amplified by Hanbio Biotechnology (Shanghai China). We selected the cell line with G418 (600 µg/mL) for 3 weeks and expanded it, and C33a cells that overexpressed CD36 were labeled “C33a–CD36” for our study.
Wound healing assay and trans-well assay
The C33a/CD36, C33a/vector (control), SiHa/siRNA, SiHa/nc-siRNA (control), HeLa/siRNA, and HeLa/nc-siRNA (control) cells were seeded into 12-well plates at a density of 1 × 105 cells/well. The cells were then scraped with a 200-μL sterile pipette tip when they formed monolayers. After washing the cells three times with PBS, we used serum-free medium for culture, and photographed the cells at 0 and 48 h.
We performed the invasion assay using transwell plates (Costar, USA). The C33a/CD36, C33a/vector, SiHa/siRNA, SiHa/nc-siRNA, HeLa/siRNA, and HeLa/nc-siRNA cells (each at a density of 1 × 105 cells/well) were added to the upper chamber with 0.2 mL of serum-free RPMI-1640; we added 0.5 mL of 10% FBS medium to the lower chamber. The cells were allowed to invade for 48 h at 37 °C. After removing the cells on the upper surface of the membrane, we stained cells on the lower aspect with trypan blue.
Colony formation assay and evaluation of cellular apoptosis by flow cytometry
We adjusted the concentrations of C33a/CD36, C33a/vector, SiHa/siRNA, SiHa/nc-siRNA, HeLa/siRNA, and HeLa/nc-siRNA cells to appropriate densities, and then inoculated each culture dish with 200 cells at 37 °C, changing the medium every 4 days. After 2 weeks, cells were stained with trypan blue, and numbers of cell colonies were counted using a light microscope.
Analysis of cellular apoptosis was conducted strictly following the instructions of the apoptosis kit (KeyGEN BioTECH, Nanjing, China). C33a/CD36, C33a/vector, SiHa/siRNA, SiHa/nc-siRNA, HeLa/siRNA, and HeLa/nc- siRNA cells were incubated with the DNA-binding dye propidium iodide (50 ug/mL) and RNase (1.0 mg/mL) for 20 min at 37 °C in the dark. We then washed the cells and analyzed the emitted red fluorescence with a flow cytometer (BD, Heidelberg, Germany).
Immunofluorescence
C33a/CD36, C33a/vector, SiHa/siRNA, SiHa/nc-siRNA, HeLa/siRNA, and HeLa/nc-siRNA cells were cultured in 24-well plates (at 1 × 105/mL) overnight. The cells were washed twice with ice-cold PBS, fixed with 4% paraformaldehyde for 20 min, and then permeabilized with 0.5% Triton X-100 for 10 min at room temperature. The samples were then incubated with primary antibodies, including CD36 (1:50 dilution) and vimentin (1:100 dilution) at 4 °C overnight. After washing 3 times with PBS, we incubated the samples with secondary antibodies (Alexa Fluor 488, Alexa Fluor 594, 1:500 dilution) mixed with DAPI (1:1000 dilution) for 1 h in the dark. After washing three times and covering the samples with anti-fluorescence quencher, we recorded the images using a fluorescence microscope.
Western immunoblotting analysis
We lysed the cells in a lysis buffer after washing twice with ice-cold PBS, and quantified total protein concentrations with a BCA kit (Beyotime Biotechnology, Guangzhou, China). Twenty micrograms of total protein was boiled for 5 min before being loaded onto 10% polyacrylamide gels and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were incubated with primary antibody, including anti-CD36 (1:200 dilution), anti-E-cadherin (1:500 dilution), anti-TGF-β (1:500 dilution), anti-vimentin (1:500 dilution), anti-snail (1:500 dilution), anti-slug (1:500 dilution), anti-twist (1:500 dilution), anti-GAPDH (1:1000 dilution), and anti-β-actin (1:1000 dilution) at 37 °C overnight. Next, the membranes were incubated with a secondary antibody for 1 h, and the specific protein bands on the membranes were detected using an enhanced chemiluminescence kit (Beyotime, China).
In vivo experiments
A nude mouse xenograft model was established using 4-week-old female BALB/C nude mice obtained from Hunan SJA Laboratory Animal Center. C33a/CD36 and C33a/vector cells (2 × 106/mL) were subcutaneously injected into the lower abdomen or tail vein of the nude mice, and the tumor diameters for each mouse were measured weekly. After 5 weeks, we euthanized the mice using anesthesia, and the tumors were removed and measured. All of the animal protocols were conducted in accordance with the Institutional Animal Ethics Care Committee.
Statistical analysis
All statistical analyses were conducted using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). We performed experiments in triplicate, and data are presented as mean ± SEM. The χ2 test was used to analyze the relationship between CD36 levels and clinicopathologic characteristics. Data from two groups were analyzed by unpaired t tests; and, if more than two groups, by one-way ANOVA. A P value of < 0.05 was considered statistically significant.