Cell lines and transient transfection
The HEC-1-B cells were purchased from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in modified Eagle’s medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). And cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2.
The lentivirus-expressed CypB-specific short hairpin RNA (shRNA) was used to knock down the expression of CypB and negative control shRNA (NC-shRNA) as control. These shRNAs were used for transfection in the HEC-1-B cell line following the manufacturer’s protocol
Sample collection
All the tissue samples were collected via biopsy of surgical resection without chemotherapy or radiotherapy between December 2017 and September 2018 in the department of Gynecology, Affiliated Yantai Yuhuangding Hospital (Yantai, Shandong, China). The study was approved by the Ethics Committee of Yantai Yuhuangding Hospital on November 27, 2017 (registration number: YLYLLS [2018] 008). All samples were collected after obtaining written informed consent. The samples were snap-frozen in liquid nitrogen and stored at −80°C before RNA extraction or generation of formalin-fixed, paraffin-embedded tissue sections for immunohistochemistry.
Cell proliferation and clone formation
Cell proliferation was determined using the Cell Counting Kit-8 assay (CCK-8; Beyotime Biotechnology, Shanghai, China) per the manufacturer's instructions. Cells in the logarithmic growth phase (1 × 104 cells/mL per well) were grown in 96-well plates in medium containing 10% FBS in an incubator with 5% CO2 at 37°C for 72h after transfection. Afterward, 10mL of CCK-8 solution was added to each well, and the plates were incubated for an additional 4h. The absorbance in each well was measured at a wavelength of 450nm with a microplate reader.
For clone formation, HEC-1-B cells were transfected with CypB-shRNA for 48h and were collected and seeded in triplicate into 6-well plates at a density of 1,000 cells/mL per well. The cells were incubated for 10 days at 37°C in a 5% CO2 atmosphere. They were then fixed with 4% paraformaldehyde for 30 min and stained with Giemsa (Beyotime Biotechnology) for 20min. After washing with double-distilled H2O several times, images of the cell plates were taken (Canon, Inc., Tokyo, Japan).
In vitro wound healing
The wound-healing assay was performed to evaluate cell migration. Cells were seeded onto 35mm dishes. After cells reached over 90% confluence, using a sterile pipette tip to make a scratch through the confluent monolayer. The medium was changed and cells were cultured for 24 hours. The percent wound closure was calculated for four randomly chosen fields.
Invasion assays
For the invasion assay, 105 cells in serum-free medium were placed into the upper chamber of the insert with Matrigel (BD Biosciences, Franklin Lakes). After 24 hours of incubation at 37°C, we removed the cells remaining in the upper chamber or on the upper membrane. The number of cells adhering to the lower membrane of the chambers was counted after staining with a solution containing 0.1% crystal violet (Beyotime Institute of Biotechnology, Beijing, China) and 20% methanol.
RNA extraction, reverse transcription, qRT-PCR, western blot, and microarray analysis
Total RNA was extracted using TRIzol, and cDNA was synthesized with the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Gene expression was assessed by qRT-PCR using SYBR Premix Dimer Eraser (Perfect Real Time, TaKaRa) assay kits. Relative fold changes in expression were calculated using the comparative Ct (2-∆∆Ct) method.
Total protein was collected from cells treated with RIPA lysis buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membrane (Millipore, Bedford, MA). Primary antibodies used in this study were shown as follows: rabbit polyclonal antibodies for CypB, Ang2 (immunoway, USA), and VEGF (Weiao, China). Beta-actin protein (Santa Cruz, CA) was used as a loading control.
The RNA samples collected 72 hours after lentivirus transfection were submitted to Phalanx Biotech (Hsinchu, Taiwan) for microarray analysis. We used the Phalanx Human OneArray Plus Gene Expression Profiling platform 6.1 to analyze the CypB-mediated alterations of mRNA expression.
Immunohistochemistry (IHC)
Sections (4µm) were cut from the constructed TMA blocks, deparaffinized, and rehydrated. Heat-induced epitope retrieval was performed onboard of the Leica Bond RX platform at 100℃ using EDTA buffer (pH 9.0, Leica) for 20minutes, followed by 15 min of incubation with anti-CYPB antibody (#43603, Cell Signaling Technology, Danvers, MA, USA) or anti-β-catenin antibody (#8480, Cell Signaling Technology) at room temperature and with Bond™ Polymer Refine Detection kit (Leica Biosystems, Buffalo Grove, IL, USA) for 8min. The reaction was visualized using 3,3′-diaminobenzidine tetrahydrochloride for 10min and with hematoxylin as a counterstain. Scoring was performed by pathologists (MK, PR) using a Nikon Eclipse microscope on TMA glass slides at 20× magnification. Tissues were scored for CypB expression, and the scoring system reflected the extent and intensity of staining: the intensity was assigned a score of 0, 1, 2, or 3, representing negative, weak, moderate, or strong expression, respectively; while the extent was assigned a score of 0, 1, 2, 3 or 4, representing <5%, 6–25%, 26–50%, 51–75% and >75% of cells stained. The overall quantitation of the score was obtained by multiplying the average intensity and score of five different high-power fields (at 400× magnification). The samples were divided into two groups based on final staining scores, which ranged from 0 to 7: the high expression group (scores of ≥4) and the low expression group (scores of <4)[26].
Gene ontology functional and pathway enrichment analysis
GO (gene ontology) and KEGG pathway enrichment analysis was used for differentially expressed genes (DEGs) using the DAVID database. FDR values of < 0.05 were set as the cut-off criterion for the two analyses.
Statistical analysis
Statistical analysis was performed using SPSS software, version 18.0 (SPSS, Chicago, IL, USA). The chi-square test was used to determine the differences in age and tumor grades between high and low expressed CypB groups. Differences between two groups were analyzed using Student’s t-test for comparison of two groups or by one-way analysis of variance for comparison of more than two groups. P values of < 0.05 were considered statistically significant.