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 CYPB-siRNA and negative control siRNA (NC-siRNA) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA; sc-35145 and sc-36869, respectively). Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.
Sample Collection
All the tissue samples were collected via biopsy of surgical resection 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 in 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 prior to 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 72 h. Afterwards, 10 mL of CCK-8 solution was added to each well, and the plates were incubated for an additional 4 h. The absorbance in each well was measured at a wavelength of 450 nm with a microplate reader.
For clone formation, HEC-1-B cells were transfected with CYPB-siRNA for 48 h 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 20 min. After washing with double-distilled H2O several times, images of the cell plates were taken (Canon, Inc., Tokyo, Japan).
RNA extraction, reverse transcription, qRT-PCR and microarray analysis
Total RNA was extracted using TRIzol, and cDNA was synthesized with 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. Purified RNA samples 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 (4 µm) 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 20 minutes, 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 8 min. The reaction was visualized using 3,3′-diaminobenzidine tetrahydrochloride for 10 min and with hematoxylin as a counterstain. Scoring was performed by pathologists (MK, PR) using 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)[23].
Gene Ontology Functional And Pathway Enrichment Analysis
GO and KEGG pathway enrichment analysis were 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.