Cell cultures
HeLa and SiHa cell lines were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Biowest, France). Cervical cancer cells were grown at 37℃ with 5% CO2 and 95% room air.
Cell transfections
Lentiviral particles harboring shRNA targeting CENPK (sh-CENPK) and negative control shRNA (sh-NC) were designed and constructed by GeneChem Corporation (Shanghai, China). Lentivirus particles were transfected into cervical cancer cells using polybrene reagent (GeneChem, Inc.). Plasmids (ov-β-catenin, ov-CENPK) were designed and constructed by Vigene Biosciences Corporation (Shandong, China). siRNAs (si-iRhom2, si-CENPK, si-SOX6, si-p53, si-RAD21, si-SMC3) were obtained from RiboBio Corporation (Guangzhou, China) (shown in Additional file 6 Table S1). Following the manufacturer’s protocol, plasmids and siRNAs were transduced into cervical cancer cells using Lipofectamine TM 2000 (Invitrogen Corporation, Shanghai, China). Forty-eight to seventy-two hours after transfection, cells were subjected to further experimentation.
Tumorsphere formation assay
The protocol of tumorsphere formation assay was in line with a previous method[16]. Briefly, cervical cancer cells (5,000/well) were seeded on 6-well ultra-low-attachment plates (Corning, Inc., NY, USA) and cultured in serum-free DMEM/F12 with 2% B27, 20 ng/ml EGF, and 20 ng/ml FGF. Fresh medium was replaced every 2 days. The tumorspheres were recorded and counted on day 14 post-seeding, and the number and size of tumorspheres were analyzed after passaging for three generations.
MTT assay
The MTT assay was used to measure cell proliferation. Cervical cancer cells (1,000 cells/well) were seeded in 96-well plates. At the indicated time, MTT (5 mg/ml; Sigma-Aldrich Corporation, MO, USA) was added to each well. After 4 h of incubation, dimethyl sulfoxide (Sigma-Aldrich Corporation, MO, USA) was added to each well. The absorbance value (OD) of each well was detected at 490 nm.
Colony-formation assay
For the measurement of cell proliferation, cervical cancer cells were plated at a density of 100 cells per well. After 10 days of culture, colonies were fixed with methanol and stained with a hematoxylin solution. The number of colonies (≥ 50 cells) was counted under a microscope. For measurement of cell chemoresistance, transfected cervical cancer cells were treated with cisplatin or carboplatin for 6 h at the indicated concentrations before seeding at a density of 500 cells per well. After 10 days of incubation, colonies were fixed, stained, and photographed for analysis.
Transwell assays
Transwell assays were applied to evaluate the migration and invasion capacity of cervical cancer cells. Cells suspension were added to the upper chamber of Transwell coated with (for invasion assay) or without (for migration) Matrigel purchased from BD corporation, and the lower chambers of Transwell were filled with Dulbecco's modified Eagle's medium containing 10% FBS. The migrated and invaded cells were fixed, stained, and photographed under microscopy.
EdU incorporation assays
Following the manufacturer’s protocol, EdU incorporation was conducted using the Apollo567 In Vitro Imaging Kit (RiboBio Corporation, Guangzhou, China). After 2 h treatment with EdU (10 μM), cervical cancer cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and co-stained with Apollo fluorescent dyes and DAPI (5μg/ml). The EdU-positive cells were photographed and counted under a microscope.
Immunofluorescence
Cervical cancer cells were seeded and grew on coverslips. After incubation, the cells were subjected to fixation with 4% paraformaldehyde, permeabilization with 0.2% Triton X-100, and incubation with antibodies (shown in Additional file 7 Table S2). The cells were then co-stained with DAPI (0.2 mg/ml) and observed under a fluorescence confocal microscope.
RT-PCR and QPCR
Total RNA was extracted from cervical cancer cells or xenografts using TRIzol (Invitrogen Corporation, Shanghai, China), and cDNA was generated using a reverse transcription reagent kit (TaKaRa Corporation, Dalian, China). Then, the synthesized cDNA was used as a template for RT-PCR and QPCR with specific primers (shown in Additional file 8 Table S3) on Bio-Rad T100 and Bio-Rad CFX 96, respectively. β-actin was used as a control. For RT-PCR, the images were visualized by Bio-Rad GelDoc XR+. For QPCR, the relative mRNA expression was quantified using the 2-ΔΔCt method.
Western blot analysis
Cervical cancer cells were lysed in lysis buffer, and proteins were quantified by the BCA method. After loading, proteins were separated, transferred, and immunoprobed with specific antibodies (shown in Additional file 7 Table S2). Chemiluminescence was used for protein detection, and images were captured using a Bio-Rad ChemiDocTM CRS+ Molecular Imager.
Animal studies
The Institutional Animal Ethical Committee, Experimental Animal Center of Fujian Medical University approved the protocols for animal studies. All experiments conform to all relevant regulatory standards. BALB/c-nu mice (4-5-week-old, female) (n = 5 per group) were subcutaneously injected with 5 × 106 cells/100 μl in the flank. The longest diameter (A) and the shortest diameter (B) of the growing tumors were measured every 3 days with a caliper, and the tumor volume (V) was calculated by the equation V = (AB2)/2. Twenty days after cell injection, the mice were euthanized and tissue was taken for further experimentation. The subcutaneous xenograft mouse model was constructed and used to evaluate tumor formation ability and tumor growth. A series of 5×105, 2×105, and 5×104 cells inoculated subcutaneously into BALB/c-nu mice (4-5-week-old, female) (n = 6 per group), and the tumor-initiating frequency was evaluated.
A pulmonary metastasis model was applied to detect cervical cancer metastatic ability. BALB/c-nu mice (4-5-week-old, female) (n = 5 per group) were injected with 1 × 106 cells/100 μl through the tail vein. Four weeks after injection, the formation of lung metastasis was evaluated under light and fluorescent microscopy.
The subcutaneous xenograft mouse model was also used to determine the chemoresistance of cervical cancer tumors. Cervical cancer cells (5 × 106 cells/100 μl) were inoculated in the flank of the BALB/c-nu mice (4-5-week-old, female) (n = 10 per group). The mice were treated with cisplatin (3 mg/kg, i.p.) once a week for six weeks[17, 18] or carboplatin (30 mg/kg, i.p.) once a week for four weeks[19]. Survival curves were plotted using Kaplan-Meier analyses.
Luciferase reporter assay
The assays of Wnt signaling activity, p53 signaling activity, and the transcriptional activity of RAD21/SMC3 on CENPK were carried out using the luciferase assay system. Cervical cancer cells were co-transfected with TOPflash or FOPflash with pRL (Millipore Corporation, MA, USA) to detect Wnt signaling. Cervical cancer cells were transfected with pGL4 luciferase reporter vector (Promega Corporation, Madison, USA) harboring p53 response element to detect p53 signaling. Cervical cancer cells were transfected with pGL4 luciferase reporter vector (Promega Corporation, Madison, USA) harboring RAD21/SMC3-binding sites in the CENPK promoter region to detect the transcriptional activity of RAD21/SMC3 on CENPK. Forty-eight hours after transfection, cells were collected and subjected to luciferase activity measurement using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, USA) on a BioTek luminometer. The relative activity of each reaction was calculated as the ratio of firefly to Renilla luciferase.
Co-immunoprecipitation (Co-IP)
According to the manufacturer’s instructions, Co-IP was conducted with a Pierce Co-Immunoprecipitation kit (Thermo Scientific, Shanghai, China). In brief, total proteins were lysed from cervical cancer cells and subjected to concentration quantification. Proteins (5 mg) were then incubated with specific antibodies or IgG, which was applied as a negative control. The enriched proteins were eluted and used for western blot analysis.
Chromatin immunoprecipitation (ChIP)
The ChIP assay was conducted using a ChIP assay kit (Thermo Scientific, Shanghai, China) according to the manufacturer’s instructions. Briefly, chromatin from cervical cancer cells was crosslinked, extracted, and clipped with Micrococcal Nuclease to generate DNA fragments. Immunoprecipitation was performed with specific antibodies (shown in Additional file 7 Table S2) or IgG, which served as a negative control. The DNA fragments were eluted, purified, and subjected to PCR and qPCR.
Cycloheximide (CHX) chase assay
Cervical cancer cells were treated with cycloheximide (50 µg/ml) at the indicated time. Subsequently, proteins were collected and quantified using a BCA method. The proteins were further subjected to western blot analysis, and signals were recorded for evaluating the half-life of the proteins.
Cell fractionation assay
The assay was carried out using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Shanghai, China). Cervical cancer cells were sequentially treated with ice-cold CER I and the CER II extraction reagent. After centrifugation, the supernatant containing cytoplasmic extract was collected. The pellet was further incubated with NER extraction reagent. After centrifugation, the supernatant containing nuclear extract was retained. The collected proteins were further subjected to western blot analysis.
Patient tissues
One hundred and nineteen paraffin-embedded cervical cancer specimens and 35 paraffin-embedded adjacent normal tissues in a tissue chip (HUteS154Su01) was purchased from Shanghai Outdo Biotech (Shanghai, China). All the patients underwent surgery and had a confirmed pathological diagnosis. Clinical data were extracted from the medical records of the patients. Some clinicopathological characteristics, such as histology grade and Ki67 status, were not available for all patients. Patient consent and ethics approval were obtained from the Ethics Committee of Shanghai Outdo Biotech Corporation.
Immunohistochemistry (IHC)
Paraffin-fixed sections (4 μm) from tissues were deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer. After eradicating endogenous peroxidase activity with 3% H2O2 and blocking non-specific antigens with goat serum, the sections were incubated with specific antibodies (shown in Additional file 7 Table S2). The signal of the sections was measured using the DAB substrate (Maixin Biotech. Corporation, Fuzhou, China). Staining intensities were evaluated as previously described[15].
Bioinformatics analysis
The mRNA-Seq data of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) in The Cancer Genome Atlas (TCGA) database were downloaded and applied for performing differential expression analysis, correlation analysis, gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA) with R software. The median of mRNA expression values was assigned as a cut-off value, and samples were classified into high- and low-expression groups according to the cut-off value.
Statistical analysis
The data are presented as the mean ± SD from at least three independent experiments and analyzed by SPSS 22.0 or RStudio. Statistical significance was detected using the Student’s two-tailed t-test for two groups, one-way ANOVA for multiple groups, and a parametric generalized linear model with random effects for the growth curve. The correlations between CENPK expression and clinical parameters were measured using the Chi-square test. The correlations among gene expressions were measured using Spearman’s rank correlation test. The Wilcoxon rank-sum test was applied to investigate the difference of CENPK expression between cancers and adjacent normal tissues. Kaplan-Meier survival curves were plotted for survival analysis, and a log-rank test was used to explore the difference. Cox regression models were adopted to identify the relationship between CENPK and the survival time of cervical cancer patients. All statistical tests were two-sided and a P value of < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01 and ***P < 0.001.