Tissue sample collection
This retrospective study was approved by the Ethics Committees of Xiangya Hospital, Central South University. Patients were fully informed of the study and provided consent before specimen collection. A total of 87 EC specimens from consecutive patients (cancer and adjacent normal tissues) were confirmed by postoperative pathological diagnosis from March 2015 to November 2015 at Xiangya Hospital Central South University. For all patients, relevant clinical data were collected and checked again for confirmation. The age of patients ranged from 31 to 70 years and mean age was 51.3 ± 12.2 years. A total of 75 cases were endometrioid adenocarcinoma, 5 were adenosquamous carcinoma, 3 were serous papillary adenocarcinoma, 2 were clear cell carcinoma, and 2 were mucinous carcinoma. According to 2009 FIGO guidelines[5] for the surgical staging of EC, 32 were Ia, 36 were Ib, 5 were II, 12 were III, and 2 were IV. Histopathological grading was as follows: 49 cases were G1, 33 cases were G2, and 5 cases were G3.
Immunohistochemistry (IHC)
Specimens were fixed in 10% formalin, embedded in paraffin, and sectioned. Two experienced pathologists made the histological diagnosis, and CCT7 expression was evaluated using SP Immunohistochemistry Kits (Abcam Ltd., Cambridge, UK). Results were evaluated according to the following criteria (where brown staining of the cytoplasm and cell membrane indicated positive results) based on the degree of positive staining: weakly positive (+), 1 point; positive (+), 2 points; strongly positive (+++), 3 points. Additionally, based on the percentage of positive cells, they were scored as follows: <25% positive cells (+), 1 point; 25% to 50% (+), 2 points; ≧50% (+++), 3 points; no positive cells indicated a negative result. Finally, a comprehensive indicator was calculated by summing the two scores, where ≦3 points was defined as negative, 4 to 6 points was interpreted as positive, and >6 points was interpreted as strongly positive.
Western blotting analysis
Proteins were isolated from EC tissues via NP-40 lysis buffer (Abcam Ltd., Cambridge, UK) and then separated in 12% SDS-PAGE gels and blotted on nitrocellulose membranes. The filters were hybridized with polyclonal anti-CCT7 (Abcam Ltd., Cambridge, UK) at 4°C overnight, followed by incubation with the secondary anti-rabbit (Abcam Ltd., Cambridge, UK) for 1 h at room temperature. Anti-Tubulin (Abcam Ltd., Cambridge, UK) were used as the loading control. Gray-scale values were analyzed by ImageJ software and also was described previously[19]
Cell culture and transfection
Endometrial cancer Ishikawa (Shanghai Zhongqiaoxinzhou Biotech, ZQ0472) and RL95-2 (Shanghai Zhongqiaoxinzhou Biotech, ZQ0362) cell lines were cultured in Dulbecco's Modified Eagle's Medium (Hyclone) supplemented with 5% fetal bovine serum (FBS, Gibco), 300 mmol/L l-glutamine (Hyclone), 5 µg/mL bovine insulin (Hyclone), 10,000 units/mL penicillin (Hyclone), and 10,000 µg/mL streptomycin (Hyclone) at 37 degree under 5% CO2. The role of CCT7 in Ishikawa and RL95-2 EC cells was examined using siRNA-mediated CCT7 knockdown. For this analysis, 50 µM, 100 µM, and 200 µM siRNA targeting CCT7 (CCT7-Homo-914: 5′-CCACACAGUUGAGGAUUAUTT-3′, 5′-AUAAUCCUCAACUGUGUGGTT-3′;CCT7-Homo-986: 5′-CCAUCAUUCUGGAGCCAAATT-3′, 5′-UUUGGCUCCAGAAUGAUGGTT-3′; or non-targeting negative control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′; Sangon Bio-technology Co. Ltd., Shanghai, China) and Dharma FECT reagent (Thermo Fisher Scientific, Waltham, MA, USA) were transfected into cells according to the manufacturer's instructions. After 48 hours, the expression of CCT7 was evaluated by qRT-PCR methods described previously[20], and the best CCT7 siRNA, i.e., the siRNA resulting in the lowest CCT7 expression level, was selected. Then, Ishikawa and RL95-2 cells were transfected with CCT7-targeting siRNA, and cell proliferation, apoptosis, cell cycle, migration and colony formation assay were examined.
Cell proliferation assay
Cell proliferation was determined using an MTT assay. Briefly, cells (5 × 103) were plated on 96-well plates for 24 hours and then cultivated for 24 hours, 48 hours, and 72 hours. By metabolic conversion of MTT dye, viable cell densities were determined. Absorbance was read at 490 nm to evaluate assay results.
Cell apoptosis assay
Cell apoptosis was determined using an Annexin V assay and described previously[21]. After transfection and/or lidocaine treatment, cells were collected, washed, and suspended in Annexin V-binding buffer. FITC-conjugated Annexin V and propidium iodide (PI; Beyotime, Haimen, China) were added to cells successively. After incubation, Annexin V-binding buffer was added, and cells were analyzed using a FAC Scan Flow Cytometer.
Cell cycle analysis
After transfection and/or lidocaine treatment, cells were harvested after trypsinization. Then, cells were rinsed three times with buffer solution, the concentration was adjusted to 1 × 106 cells/mL, and the Cycle TEST PLUS DNA Reagent Kit (Becton Dickinson, Franklin Lakes, NJ, USA) was used according to the manufacturer’s instructions. Cell cycle status was analyzed by flow cytometry using PI. The PI fluorescence intensity of 10,000 cells was measured for each sample.
Cell migration assay
Ishikawa and RL95-2 cells were treated with 0.25% trypsin and suspended in serum-free medium. Ten thousand cells were added to each Transwell and serum-free medium was added to reach 100 µL. The lower chamber was supplemented with 10% fetal bovine serum and 1640 medium, followed by cultivation for 48 hours in an incubator. The broth in each well was discarded, and cells were washed twice with phosphate-buffered saline (PBS). The surfaces of cells were wiped with wet cotton and fixed with acetone: methanol (1:1) at room temperature for 20 minutes. After cells were washed twice with PBS, they were stained for 15 minutes with 0.1% crystal violet and washed with PBS three times or more. Finally, images were obtained under an inverted microscope. Absorbance was read at 550 nm to evaluate assay results.
Cell colony formation assay
As described previously[22], Ishikawa and RL95-2 cells in logarithmic growth phase were treated with 0.25% trypsin and suspended in 10% FBS medium. Then, cells were seeded in 6 well plate with 1ml complete medium (500 cells per well) and cultured at 37 degree under 5% CO2 for 2-3 weeks until eye-visible cells colony. After washing twice with PBS, fixing cells with 1ml 4% paraformaldehyde, and then staining with 0.5% crystal violet for 30mins. Remove the dye with flow water and dry in air, obtain the image and count the cells colony.
Bioinformation analysis
The CCT7 expression data of UCEC (Uterine Corpus Endometrial Carcinoma) and normal tissue were carefully downloaded from the TCGA Data Portal website (http://cancergenome.nih.gov). Co-expressed genes of CCT7 in UCEC were collected from MEM (http://biit.cs.ut.ee/mem), UALCAN (http://ualcan.path.uab.edu), and GEPIA (http://gepia.cancer-pku.cn) for further evaluation. Gathered genes were analyzed using bioinformatics. The enrichment of functions and signaling pathways of the target genes were analyzed in Enrichr (https://amp.pharm.mssm.edu/Enrichr/). The String database (http://www.string-db.org) was applied to construct the protein-protein interaction (PPI) network for the hub gene identification. Moreover, hub genes were selected to obtain their expression and correlation with CCT7 in UCEC.
Statistical analysis
All results are expressed as means ± standard deviation (SD) from three independent experiments. The Chi-square test was used to compare positive staining rates between subgroups, and the One-way and Two-way ANOVA analysis of variance were used to compare other data implemented in SPSS 18.0. GraphPad Prism was applied to acquire the figure and receiver operating characteristic curves (ROCs). P < 0.05 was considered statistically significant.