Patients and tissue specimens
Sixty-two patients and surgical tissue samples were selected from among patients with ESCC who underwent radical operation in the thoracic surgery department of Tongji Hospital of Shanghai From January 1, 2010, to December 31, 2015. All patients were followed up during the study period. The study was conducted according to the Declaration of Helsinki (as revised in 2013) and was approved by the ethics committee of Tongji Hospital Affiliated to Tongji University. Before participating in this study, each patient signed an informed consent form.
Bioinformatics Analysis
The pan cancer analysis used RNAseq data from TCGA and GTEx databases. Data in TPM (scripts per million reads) format is obtained from UCSC XENA database (https://xenabrowser.net/datapages/ ) and be handled through the Toil[25] process. The subgroup analysis of esophageal cancer was completed by using the online tool of ualcan (http://ualcan.path.uab.edu/index.html ). GSEA (Gene Set Enrichment Analysis) and GO (Gene Ontology) function annotation were performed using R (4.2.0 version) package “ clusterProfiler”. The Hallmarks gene set in the gene set database MSigDB Collections was used for GSEA analysis. According to the list of given genes based on a large number of genomics and proteomics data, we used the GeneMANIA database (http://www.genemania.org) to predict the 50 most relevant genes of NEK2-E2F1-IGF2 regulatory axis.
Cell lines and cell culture
In this study, human esophageal cancer cell lines (TE1, TE5, KYSE-410, KYSE-150, Eca-109) and esophageal epithelial cells (Het-1A) were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in medium containing 10% serum and penicillin/streptomycin liquid (penicillin 100 U/ml, streptomycin 100 µg/ml). The culture medium consisted of 90% RPMI 1640 and 10% FBS (Gibco, USA). All cells were cultured in the incubator with 95% O2 and 5% CO2 at 37 ℃. NEK2 shRNA was purchased from Shanghai Jikai company. The sequence of the shRNA for NEK2 (shNEK2; from 5 'to 3') was as follows: AGACGAGCAAAGAAGAAAT.
Antibodies
An anti-NEK2 antibody (ab227958, Abcam, USA), an anti-GAPDH antibody (ab181602, Abcam, USA), a recombinant anti-E2F1 antibody (ab179445, Abcam, USA), and an anti-IGF2 antibody (ab177467, Abcam, USA) were used in the current study. The related reagents included the proteasome inhibitor MG132 ( Z-Leu-Leu-Leu-al) (MedChemExpress, USA) and the protein synthesis inhibitor cycloheximide (CHX) (Abcam, USA).
Immunohistochemistry
Paraffin sections were immersed in xylene for dewaxing, hydrated through a graded ethanol series, and then placed into EDTA repair solution (pH = 9.0). The antigens were repaired via heating in a water bath at 100 ℃ for 15 minutes. Hydrogen peroxide (3%) was added to the sections to block endogenous peroxidase. The sections were blocked in serum and incubated overnight with primary antibodies at 4 ℃. The sections were incubated with horseradish peroxidase-labeled second antibodies the next day. The staining was developed with diaminobenzidine (DAB). After restaining with hematoxylin, the slices were dehydrated with graded ethanol and fixed with neutral gum. Immunohistochemical staining intensity was scored according to the following scale: 0, negative; 1, weakly positive; 2, moderately positive; and 3, strongly positive. The positive cell frequency was scored as follows: 0, < 5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; and 4, > 75%. In data analysis, a total score of 0–7 was considered low expression, and a score of 8–12 was considered high expression.
Quantitative real time-PCR
Knockdown efficiency in tumor cells was tested by quantitative real-time PCR. First, RNA was extracted from ESCC cells by the TRIzol method (Ambion). The optical density (OD) value was determined with a micro spectrophotometer, and the RNA concentration was calculated. The RNA was stored in a freezer at -80 ℃ until use. The extracted RNA was reverse-transcribed into cDNA with a PrimeScript™ RT Reagent Kit with gDNA Eraser, and the cDNA was amplified by quantitative real-time PCR using TB Green Premix Ex Taq™ II (Takara). GAPDH was used as an internal reference to reduce the experimental error. The primers for qRT-PCR are described in Annex 1.
Western blotting
Cells were placed in a mixture of RIPA (Epizyme, China) and PMSF. After centrifugation, the supernatant was obtained, and the protein concentration was determined by BCA method. Protein samples were separated via 10% SDS-PAGE. After electrophoresis, the proteins in the gel were transferred onto a PVDF membrane. The PVDF membrane was incubated with a mixture of primary antibodies and primary antibody buffer at 4 ℃ overnight. On the second day, it was incubated with secondary antibodies for 1 hour to develop. All Western blot reagents were purchased from Epizyme.
CCK-8 cell proliferation assay
Cell proliferation was detected by using a CCK-8 (Dojindo Laboratories, Japan). First, cells were cultured in 96-well plates at a density of 5000 cells per well in a mixture of 10% serum and 90% RPMI-1640 medium. After the indicated culture duration, 10 µl of CCK-8 solution was added to each well, and the plates were incubated in an incubator for 2 hours. The absorbance at 450 nm was measured with an ELISA reader.
EdU cell proliferation assay
Cells in the logarithmic growth phase were inoculated into 96-well plates at a density of 4 × 103 cells per well and cultured to the normal growth stage. The cells were incubated in 50 µM EdU medium (RiboBio, Guangzhou, China) for 6 hours. Then, the cells were fixed and stained with EdU. The cells were restained with Hoechst 33342 reaction solution and observed under a fluorescence microscope.
Colony formation assay
Cells were seeded on a 6-well plate at a density of 400 cells per well. After 15 days of culture in a 5% CO2 incubator at 37 ℃, the cells were washed with PBS and stained with 0.1% crystal violet. The colony formation was observed under a microscope.
Wound healing assay
Cells were seeded on a 6-well plate at a density of 5 × 105 cells per well and cultured in a 5% CO2 incubator at 37 ℃. After they reached confluence, the cells were scratched with a pipette ,then washed with PBS three times, and cultured in serum-free medium for 24 hours. Finally, the cells were observed under a microscope, sampled and photographed.
Migration and invasion assays
Transwell chambers (Corning, USA) were used for migration and invasion experiments. In the migration experiment, 5×104 cells were placed in the upper compartment with 1% FBS and RPMI-1640 medium. In the invasion experiment, 5×104 cells suspended in 1% FBS and RPMI 1640 were inoculated into the upper compartment, which was coated with 300 µg/ml Matrigel (BD Biosciences, USA). RPMI-1640 medium containing 10% FBS was placed in the lower compartment. The cells in the upper chamber were wiped off. The cells in the lower compartment were fixed with methanol for 10 minutes and then stained with crystal violet. The cells were counted and imaged under a microscope.
Flow cytometry
The flow cytometry experiments included a cell cycle test and an apoptosis test. A FITC/PI Kit (Kaiji, Nanjing) was used to detect apoptosis. After cell culture, the supernatant was removed, and the cells were resuspended with PBS. Binding buffer (500 µl), annexin V-FITC (5 µl) and PI (5 µl) were added in turn. In the cell cycle experiment, the digested and centrifuged cells were fixed with 80% ethanol, incubated at 37 ℃ for 30 minute(s) after addition of 100 µl of RNase (50 µg/ml). PI (400 µg, 50 ug/ml) was then added, after which the cells were stained at 4 ℃ for 30 min and detected by flow cytometry (Beckman).
Xenograft animal models
Twelve female Balb/c nude mice aged 3–4 weeks were purchased from Shanghai Jiesijie Experimental Animal Company (Shanghai, China). The nude mice were randomly divided into two groups: the TE1-negative control (NC) group and the TE1-NEK2-knockdown (KD) group. ESCC cells (5 × 106) were subcutaneously injected into the right armpits of nude mice under lidocaine local anesthesia. The longest diameter of each tumor was measured every week. At the fourth week, the nude mice were killed, and the tumor tissue was taken out for measurement. The animal experiments were approved by the Animal Committee of Tongji Hospital Affiliated to Tongji University.The maximum tumor size/burden allowed by ethics shall not exceed 2000 mm3.The largest tumor size did not exceed 2000 mm3 in this study.All animal care and procedures shall be carried out in accordance with the "Regulations on the Administration of Experimental Animals" approved by the State Council of the People's Republic of China.
Statistical analysis
All the statistics were analyzed with GraphPad Prism and SPSS 22. The paired samples were compared by Student's T-test, the measurement data of unknown distributions were compared by rank sum test, and the count data were compared between groups by the exact probability method. P < 0.05 was considered to indicate statistical significance (* P < 0.05, * * P < 0.01,***P < 0.001). All the experiments were repeated three times.