Clinical samples and TCGA database analysis
TCGA database of OV w ere used to analyze gene expression and survival rate. Twenty fresh samples with adjacent normal tissues were obtained from surgical cases. The fresh tissues were used to detect the expression of HOXB4 in OV. All the patients were informed.
Cell culture
Human OV cells of SKOV3 and OVCAR3 were obtained from Shanghai Institute of Cell Biology (Cat.TCHu185& TCHu228, Shanghai, China). Cells were maintained in DMEM (Gibco, USA) with 10% FBS (Gibco, USA) at 37 °C and 5% CO2.
RNA interference
shRNAs targeting HOXB4 were purchased from Origene Biotechnology Company (Beijing, China). The Interference efficiency of shRNA was detected by Western blot after transfected for 48h.
Colony formation assay
Cells were seeded in a six-well plate at a final concentration of 100 cells/well. After cultured for 15 days, then the cells were fixed and stained with 0.5% crystal violet (Sigma, USA). Photographed and record the number of clones greater than 50 cells
Invasion assay
8 μm pore size transwell inserts (Millipore, USA) were used to detect cell invasion ability. Cells were added to the upper insert chamber and supplement with serum-free DMEM, and the lower culture chamber was filled with DMEM containing 20% FBS. 36 h later, After the cells in the upper chamber are removed, the remaining invading cells are fixed and stained with crystal violet. The number of cells was counted under a light microscope (Nikon, Japan).
Migration assay
OVCAR3 and SKOV3 cells were seeded in 24-well plate and cultured for 24 h. The linear wound was created and washed with PBS for 3 times. Then complete medium was added and culture for 36 h. Finally, take pictures at 0, 18, and 36 hours and record the scratched area.
Western blot analysis
Total protein harvest from cells and tumor samples were separated through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Then membranes were blocked with 5% skimmed milk and incubated with the following specific primary antibodies at 4 °C overnight: anti-HOXB4 antibody, anti-E-cadherin, Vimentin, Snal1, and Zeb1 antibody. GAPDH was used as loading control. After washed with PBST, the membranes were incubated with HRP labeled secondary antibodies (Sigma,USA). Protein intensity was detected by Image Lab (Bio-Rad, USA).
qRT- PCR
Total RNA was extracted using TRIzol (Takara, Japan) in accordance with the manufacturer’s instruction. Then, 5ug RNA was used to synthesize cDNA. Using these cDNA as template, the mRNA expression of the target gene was analyzed after 30 cycles of amplification. GAPDH was used as loading control.
Luciferase reporter assay
The DHDDS motifs were amplified from human genomic DNA and cloned into a pGL4.3 luciferase reporter vector (Promega). Transactivation assays were performed using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activities were measured using a Synergy 2 microplate reader system (Gene).
Zymography assays
All media were collected and subjected to SDS-PAGE with 0.01% wt/vol gelatin. After electrophoresis, gels were stained with Coomassie R250 and destained until the wash became clear with apparent cleared zones associated with the MMP activity.
Xenograft model
In order to verify whether the effect of HOXB4 in animals is consistent with the results of in vitro experiments. A total of 18 6-week-old balb/c nude mice were purchased from Vital River (Beijing, China) and randomly divided into 3 groups. OVCAR3/nc, OVCAR3/HOXB4, and OVCAR3/DHDDS (1×106) cells were injected subcutaneously or in the tail vein. All animals were euthanized by intravenous injection of barbiturate at a final concentration of 100 mg/kg, then the tumors were removed and fixed in paraffin for further analysis. The tumor volume was calculated as follows: tumor volume = length × width2/2. All procedures performed in studies involving animals were in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC) at West China Second University Hospital.
Histology and immunohistochemistry (IHC)
Tumor tissue from nude mice were embedded and cut into 4 μm-thick sections. After microwave oven 3% H2O2 treatment, primary antibodies were added, HOXB4 antibody (1:500; Abcam, UK), MMP2 antibody (1:500; Abcam, UK), MMP9 antibody (1:300; Abcam, UK), E-cadherin antibody (1:500; Abcam, UK), vimentin, (1:500; Abcam, UK) at 4 °C overnight. The immunohistochemical staining results were collected and scored by professionals.
Statistical analysis
Statistical analyses were performed using SPSS 21.0 (SPSS Inc., USA). Statistically significant differences were analyzed using Student t test, one-way ANOVA. Differences were considered significant at P < 0.05 and labeled with *.