Clinical samples and cell culture
The tissue microarray consisting 75 NSCLC tissues and 51 adjacent normal lung tissueswere abstained fromShanghai Outdo Biotech Co., Ltd (HLug-Squ150Sur-02, Shanghai, China).
Humam NSCLC cell-lines A549, NCI-H1299, SPC-A-1and normal lung epithelial cell line EBC-1 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in RPMI-1640 (Corning Company, NY, USA) with 10% fetal bovine serum (FBS, Ausbian, Vian-Saga Biotechnology Co., Ltd, Shanghai, China) in moist air containing 5% CO2 at 37°C.
Bioinformatics analysis
The transcriptional level of CENPO between cancerous and paired normaltissuesand its relationship between clinicopathologic features was analyzed by onlineUALCAN (http://ualcan.path.uab.edu/) which based on TCGA database.(10) The prognostic role of CENPO on LUAD and LUSC was explored by Kaplan-Meier Plotter (KM Plotter, http://kmplot.com/analysis/).(11)
Immunohistochemical (IHC) staining
The paraffin sections were dewaxed in xylene, and then Antigen Retrieval Buffer (Maixin biotechnology co. LTD, Fuzhou, China). The sections were washed 3 times with 1 ×PBST for 5 min each time. 3% H2O2 was used for sealing the endogenous peroxidaseof tissues. Then sectionswere incubated with primary antibodies (anti-CENPO1:50,biorbyt), Ki67 (1:200, abcam) overnight at 4°C. After being washed, the sections wereincubated with second antibodyIgG H&L (HRP) (1:400, abcam)overnight at 4°C.The sections was stained with DAB and then counterstained with hematoxylin (Baso Diagnostics Inc.,Zhuhai, China) . Absolute ethanol was used to dehydrate the sections and then neutral resin (China National Pharmaceutical Group Co., Ltd, Beijing, China) was used to seal the sections.Based on the proportion of positive cells andthe intensity of staining, a scoring system was used to determine the expression level of CENPO, The percentages of positively stained cells were scored as follows: 0 (0%), 1 (1-25%), 2 (25–50%),3 (50-75%) and 4(≥75%). The signal intensity was analyzed as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). A final score was calculated by multiplying the percentage and signal intensity scores.The specimen with final score>2 wereconsiderate as high CENPO expression, otherwise were regarded as low.
Real-time quantitative PCR (qRT-PCR)
Total RNA was extracted from cells using Trizolreagent (sigma), then RNA was reverse transcribed into cDNA by Hiscript QRT supermix for qPCR (+gDNA WIPER) (vazyme) according to manufacturer’s instruction. Two-step Real time PCR was applied. The primer sequences for qRT-PCR were as following: CENPO forward, 5′-TGCTTTTGAGGGGAACCTATTG-3′and reverse, 5′-GGGGAATGAAGACTGGGACT-3′; GAPDH forward 5′-TGACTTCAACAGCGACACCCA -3′ and reverse,5′-CACCCTGTTGCTGTAGCCAAA-3′. Total PCR system was 10uL.The relative expression level of CENPO was calculated using the 2-∆∆Ct method.(12)
Western blot
The protein were extracted from cells by 1×Lysis buffer, then the concentration was measured by BCA Protein Assay Kit (Cat 23225, Hyclone-Pierce).10% SDS-PAGE was used to separate protein and then the proteins were transferred to PVDF membrane. The membrane was blocked with1×TBST solution with 5% skimmed milk for 1 h at room temperature and then incubated at 4˚C overnight with primary antibodies. After that, the membrane was incubated with secondary antibodies at room temperature for 2 h. At last, immobilon western Chemiluminescent HRP Substrote was used to visualize the protein expression level.
Lentivirus RNAi construction and infection
Oligonucleotides for small hairpin RNAs (shRNAs) targeting the CENPO were designed (shRNA1: 5’-AGAAGCATTGGAAGAGAAATT-3’, shRNA2:5’-GCAGAGAAACCCACTGTGTAA-3’, and shRNA3:5’-CCTGGAAGAGATAGCTGCAAA-3’). The control shRNA sequence was 5’-TTCTCCGAACGTGTCACGT-3’.These pairs of oligonucleotides were annealed and cloned intoBR-V108 Vector. The generated plasmids were cotransfected into 293T cells using lipofectamine 3000 (Invitrogen, Carlsbad, USA), respectively, to generate different types of recombinant lentivirus virions. NSCLC cells were infected with shRNA-expressing recombinant lentivirus and treated with 1 lg/ml puromycin to generate CENPO-stably silencing cells. Fluorescence and infection efficiency were observed under microscope and the expression of CENPO were determined by Western blot.
Celigo cell counting assay
The cells were seeded in wells of 96-well plate (1000 cells/well,3 replicates for each group) at 37°C in an atmosphere of 5% CO2. The Celigo Imaging Cytometer (Nexcelom, Lawrence, MA, USA) was used to calculate the living cells by measuring green fluorescence in each plate. The measurementbeganafter 24 h incubation and daily measured for 5 days so that the cell growth curves were drawn.
Wound-healing assay
The cells were seeded in wells of 96-well plate at the densityof 50,000 cell/well. After the 24h, cells were scratched with pipette tips, and the ablated cells were washed away with phosphate-buffered saline (PBS). According to the degree of wound-healing, the appropriate timewas chosen to analyze the wound-healingarea by cellomics (ArrayScan VT1, Thermo, USA). The mobility of the cells was determined by measuring the wound-healingarea.
Transwell assay
Cell invasion ability was determined by Transwell assay, a Matrigel-coated modified Boyden chamber with a polycarbonate filter with a pore size of 8 μM. 100uL cell suspensions (100,000~200,000 cell) with serum-free medium was added in the upper chamber of the transwell coated with 50 mg/ml of Matrigel solution. High-serummedium (30% FBS+1640) was then added to the lower chamber. After incubation for 24 h, the cells were fixed with a 4% formaldehyde solution for 30 mins and stained with giemsa solution. Five random fields in each chamber and photographed with a microscope at 100× magnification. ImagePro +6.0 was used to calculate the average migrated cells to determine the invasion capacity.
Flow cytometry (FCM)
The impact of CENPO expression on cell cycle and apoptosis was evaluated by flow cytometry (FCM) assay. The cells were seeded in wells of 6-well plate at 37°C in an atmosphere of 5% CO2. When the cells grew to 80% confluence, they were collected by centrifugation at 1500 rpm for 5 min at 4°C and washed three times by cooled PBS solution. The cells were stained with propidium iodide (PI) (Sigma P4170) or Annexin V (eBioscience) to determine cell cycle distribution and cellular apoptosis by flow cytometry using a Guava easy Cyte HT flow cytometer (Millipore).
Human Apoptosis Antibody Array analysis
Human Apoptosis Antibody Array (Abcam, Cambridge, MA, USA) was used to detect the effect of CENPO knockdown on the apoptosis pathway-related protein expression. NCI-H1299 cells infected with shCENPO were cleaved with 2 × cell lysis buffer. The membranes were incubated with 1.2 mL cell lysate overnight at 4°C, and then the liquid was discarded. Membranes were washed with 2 mL Wash Buffer I 3 times, and 2mL Wash Buffer II 2 times, each time for 5min. 1mL 1 × Bolin-conjugated Anti-Cytokins were added and incubated with membranes overnight at 4°C. Wash Buffer I and Wash Buffer II were used to wash the membranes according to the above steps. The equal volume detection buffer C and detection buffer D were mixed evenly and added to the membranes for incubation for 2 min at room temperature. The signals were detected by chemiluminescence imaging system. And unpaired t-test was used for statistical analysis.
Nude mouse tumor formation model
Four-week old female BALB/c nude mice (Charles River Laboratories, Beijing, China) were divided into shCtrl (infected with shRNA control) and shCENPO (infected with CENPOsiRNA), 10 mice in each group.2 × 106 cells were injected intoeach nude mice. Tumor sizes were measured every 5 days using a Vernier caliper. Tumor volume was calculated using the following formula: volume = 0.5 * width2 * Length. After 39 days, the mice were sacrificed andwere placed under the in vivo imaging system (LB983, Berthold Technologies) toobserve the fluorescence. The tumors were removed from the mice, and thenthe weight of the tumors was measured. Tumor tissues were frozen in liquid nitrogen and stored at -80°C for further analysis.
Statistical analysis
Statistical analysis was carried out using SPSS software (version 21, SPSS Inc., Chicago, IL, USA). The data are presented as the mean ± S.E.M. Statistical analysis was performed with Student’s t-test or one-way ANOVA, followed by Dunnett’s test. All statistical tests were two-sided, and differences were considered to be significant at P≤0.05.