Clinical samples and TCGA database analysis
TCGA (The Cancer Genome Atlas) datasets for OV were used to analyze gene expression and survival rate. Twenty fresh samples with adjacent normal tissues were obtained from surgical cases. Fresh tissues were used to detect the expression of HOXB4 in OV. All patients were informed.
Gene annotation and enrichment analysis.
We used Metascape (metascape.org) for gene enrichment analysis of HOXB4. Metascape is an online bioinformatics pipeline with multiple gene lists that supports effective data-driven gene prioritization decisions.
The human OV cell lines SKOV3 and OVCAR3 were obtained from Shanghai Institute of Cell Biology (Cat. TCHu185 and TCHu228, Shanghai, China). Cells were maintained in DMEM (dulbecco's modified eagle medium) (Gibco, USA) with 10% FBS (fetal bovine serum) (Gibco, USA) at 37 °C and 5% CO2.
shRNAs targeting HOXB4 were purchased from Origene Biotechnology Company (Beijing, China). The interference efficiency of shRNAs was detected by Western blot after transfection for 48 h.
Colony formation assay
A colony formation assay was performed to analyze cell proliferation. Cells were seeded in a six-well plate at a final concentration of 100 cells/well. After culturing for 15 days, the cells were fixed and stained with 0.5% crystal violet (Sigma, USA). Colonies with more than 50 cells were imaged and counted.
Transwell inserts with (8 µm pore size, Millipore, USA) were used to detect cell invasion ability. Cells were added to the upper insert chamber and cultured with serum-free DMEM, and the lower culture chamber was filled with DMEM containing 20% FBS. Thirty-six hours later, after the cells in the upper chamber were removed, the remaining invading cells were fixed and stained with crystal violet. The number of cells was counted under a light microscope (Nikon, Japan).
OVCAR3 and SKOV3 cells were seeded in 24-well plates and cultured for 24 h. A linear wound was created, and the cells were washed with PBS 3 times. Then, complete medium was added and cultured for 36 h. Finally, images were taken at 0, 18, and 36 h, and the scratched area was recorded.
Western blot analysis
Total proteins harvested from cells and tumor samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF (Polyvinylidene Fluoride) membranes. Then, the membranes were blocked with 5% skimmed milk and incubated with the following specific primary antibodies at 4°C overnight: anti-HOXB4, anti-E-cadherin, anti-Vimentin, anti-Snai1, and anti-Zeb1 antibody. GAPDH was used as a loading control. After washing with PBST, the membranes were incubated with HRP-labeled secondary antibodies (Sigma, USA). Protein intensity was detected by Image Lab (Bio-Rad, USA).
ChIP-seq data analysis
The ChIP-seq data were downloaded from Cistrome Data (http://dc2.cistrome.org/#/). To verify the genes that HOXB4 binds to and their corresponding motifs, we used ChIPseeker to analyze the downloaded data according to the R package and method provided by YuLab .
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).
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 MMP (matrix metallopeptidase 2) activity.
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 4 groups: OVCAR3/nc, OVCAR3/HOXB4 (OVCAR3 cells stably expressing HOXB4), OVCAR3/DHDDS (OVCAR3 cells stably expressing DHDDS) and OVCAR3/HOXB4+siDHDDS (mice stably expressing HOXB4 were treated with DHDDS siRNA after tumor formation). A total of 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, and 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 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 was embedded and cut into 4 µm-thick sections. After microwave oven/3% H2O2 treatment, the following primary antibodies were added: anti-HOXB4 antibody (1:500; Abcam, UK), anti-MMP2 antibody (1:500; Abcam, UK), anti-MMP9 antibody (1:300; Abcam, UK), anti-E-cadherin antibody (1:500; Abcam, UK), and anti-vimentin antibody (1:500; Abcam, UK) at 4 °C overnight. The immunohistochemical staining results were collected and scored by professionals.
Statistical analyses were performed using SPSS 21.0 (SPSS Inc., USA). Statistically significant differences were analyzed using Student’s t-test and one-way ANOVA. Differences were considered significant at P < 0.05 and labeled with *.