Cells and materials
HCC1937 and MDA-MB-231 cell lines were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Genview (Campbellfield, VIC, Australia); SYBR Master Mixture was purchased from Takara (Shimogyo-ku, Kyoto, Japan); antibodies against CCT3, CDH1, Slug, Snail, VIM, mTOR, ERK1/2, p-ERK1/2, p-AKT1, P38, p-P38, NFκB-65, p-NFκB-65, β-catenin and p-β-catenin were purchased from Cell Signaling Technology (Danvers, MA, USA); and antibodies against CDH2, MMP2, FN1, MYC, p-mTOR and AKT1 were purchased from Abcam (Cambridge, MA, USA). GAPDH antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
HCC1937 and MDA-MB-231 cell lines were cultured in RPMI 1640 medium(Gibco; Gaithersburg, MD, United States) supplemented with 10% foetal bovine serum. The cells were maintained at 37°C in a 5% CO2 humidified incubator.
Lentiviral vector construction and transduction
For the construction of shRNA expression plasmids, shCCT3 was designed based on the target sequence 5’-CAAGTCCATGATCGAAATT-3’. Then, the single strand DNA oligo containing the interference sequence was synthesized, and the double strand DNA was produced by annealing. Then, the two ends of the oligo were directly linked to the lentiviral vector after enzyme digestion. The ligated products were transferred into the prepared Escherichia coli cells. Then, the positive recombinants were identified by PCR and sequenced for verification and plasmid extraction. Lentiviral vector DNA and packaging vectors were transfected into 293T cells. The empty GV115 lentiviral vector was used as the shRNA control (shCtrl). After 48 h of culture, supernatants containing lentiviruses, including shCCT3 and shCtrl, were harvested and purified. Lentiviral transduction was performed on cells at 80% confluency, with a multiplicity of infection (MOI) of 10. Seventy-two hours after infection, the cells were used for downstream assays or transplantation.
Total RNA was isolated by the TRIzol method. The cDNA reverse-transcribed from 250 ng of total RNA was amplified using the following primer sets: CCT3: forward, 5’-TCA GTC GGT GGT CAT CTT TGG-3’, reverse, 5’-CCT CCA GGT ATC TTT TCC ACT CT-3’; and GAPDH: forward, 5’-TGA CTT CAA CAG CGA CAC CCA-3’, reverse, 5’-CAC CCT GTT GCT GTA GCC AAA-3’. Real-time PCR using the SYBR Green PCR Master Mix kit was performed with an ABI Prisma 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Data were normalized to the respective GAPDH values. The value of cells infected with shControl(shCtrl) was set to 100% in each run.
Celigo image cytometry assay
The cells were trypsinized in the logarithmic growth phase, resuspended in medium, seeded into 96-well plates at 2×103 cells (100 μl) per well, and incubated overnight. A Celigo Image Cytometer (Nexcelom Bioscience, Lawrence, MA, USA) was used to evaluate the number of cells by scanning for green fluorescence daily for 5 consecutive days at room temperature. The cell proliferation curve was plotted according to the number of cells with green fluorescence.
HCC1937 and MDA-MB-231 cells infected with shCCT3 or shCtrl were seeded into 96-well plates at 1.5×103 cells and 2×103 cells per well, respectively, and incubated overnight. The cells were cultured for 5 days at 37°C. MTT assays were carried out at different time points: 24 h, 48 h, 72 h, 96 h and 120 h. Then, 20 μl MTT solution (5 mg/ml) was added to each well and incubated for an additional 4 h at 37°C. Then, the MTT solution was aspirated, and 100 μl DMSO was added to dissolve the formazan crystals. The number of cells was counted using a microplate reader at a wavelength of 490 nm.
Transwell migration assay
HCC1937 and MDA-MB-231 cells infected with shCCT3 or the shCtrl were seeded on Transwell inserts (6.5 mm, 8 μm pores) coated with or without Matrigel in 24-well plates at 60×103 cells and 80×103 cells per well, respectively, and then placed in the incubator to culture for 24 h. Cells on the upper side of the insert were scraped away, and then the inserts were fixed and stained. Invaded cells were counted under an inverted microscope.
Flow cytometry analysis
The cells were seeded in 6-well plates for apoptosis analysis or a 6-cm dish for cell cycle analysis. The cells were trypsinized at 70-80% confluency, suspended and washed with D-Hanks solution. For apoptosis analysis, cells were resuspended and stained with annexin V-APC. For cell cycle analysis, cells were fixed with 75% EtOH at -20°C for at least 2 h and then harvested and stained with PI (10 ng/ml) and RNase (10 ng/ml). Then, the cells were submitted to flow cytometry analysis.
Western blot analysis
The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime, Shanghai, China) and then transferred to polyvinylidene ﬂuoride membranes (Millipore, Billerica, MA, USA), which were blocked for 2 h with 5% nonfat milk. The membranes were incubated with primary antibodies against CCT3 (1:300), CDH1 (1:200), CDH2 (1:1000), Slug (1:1000), MMP2 (1:200), FN1 (1:300), Snail (1:1000), MYC (1:500), VIM (1:1000), ERK1/2 (1:1000), p-ERK1/2 (1:1000), AKT1 (1:1000), p-AKT (1:2000), β-catenin (1:1000), p-β-catenin (1:1000), mTOR (1:1000), p-mTOR (1:2000), NFκB-65 (1:1000), p-NFκB-65 (1:1000), P38 (1:1000), p-P38 (1:1000), and GAPDH (1:2000) overnight at 4°C. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) for 1 h at room temperature. The blots were visualized using the Super Signal West Femto kit (Pierce Biotechnology, Rockford, IL, USA).
All statistical analyses were performed using SPSS 19.0 software (SPSS, Chicago, IL, USA). Data are represented as the mean ± standard deviation. All experiments were performed in triplicate. Student’s t-test or one-way analysis of variance (ANOVA) was used for statistical analysis. For ANOVA, when a significant difference was apparent, Dunnett’s post hoc test was used to compare the means of multiple experimental groups. Differences with p < 0.05 were considered statistically significant.