Cell lines and cell culture
The human NSCLC cell lines H1299, PC-9, A549, H1975, H358, H460 and H292 and human embryonic kidney 293T cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). All cell lines were cultured according to ATCC protocols in DMEM or RPMI1640 supplemented with 10% fetal bovine serum (FBS) (Biowest, South America origin), 100 ug/ml penicillin (Sigma-Aldrich, UA), and 100 µg/ml streptomycin (Sigma-Aldrich, UA) at 37°C in a humidified incubator under 5% carbon dioxide. These cell lines were mycoplasma-free and authenticated by quality examinations of morphology and growth profile.
We performed the ONCOMINE database combined with GEO database to screen NSCLC-associated NTRs. The expression data for 24 NTRs were available from ONCOMINE. GEO datasets could be downloaded from NCBI. The survivals for the screened genes were analyzed in 117 lung adenocarcinoma patients (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13213) and 293 lung tumor samples (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30219). 503 lung adenocarcinoma patients from TCGA cohort (https://cancergenome.nih.gov/) was used to analyze the correlation between CSE1L mRNA levels and NSCLC clinicopathological features.
Human clinical specimens
Fresh human non-small cell lung cancer tissues and matched adjacent noncancerous tissues for real-time PCR and Western blot analyses were collected from the Department of Cancer at Huashan Hospital affiliated with Fudan University, Shanghai, China between 2011 and 2018. During the operation, human surgical specimens were immediately frozen in liquid nitrogen and stored at -80°C for further investigation. All of the tissue specimens for this study were obtained with patient informed consent. The study was approved by the Ethics Committee of the Ethics Committee of Fudan University.
Two tissue microarrays containing 75 and 25 paired NSCLC tissues and matched adjacent noncancerous tissues was purchased from Shanghai Biochip Co. Ltd. (Shanghai, China). Immunohistochemical staining was performed to detect the protein expression level of CSE1L in NSCLC tissues and matched noncancerous tissues. The average gray value of the image was used as a quantitative evaluation of the expression level using Image-Pro Plus 6.0 software. The proportion of the stained cells and the extent of the staining were used as the criteria for evaluation. The percentage of positive cells was scored as: < 5% (0), 5%-25% (1), 25%-50% (2), 50%-75% (3), and > 75% (4). The intensity of staining was scored as: no staining (0), light brown (1), brown (2), and dark brown (3). The final IHC scores was obtained using the traditional scoring method. IHC scores were calculated as the product of intensity (0 to 3) and the percentage of positive cells (0 to 4), yielding a range from 0 to 12.
RNA extraction and real-time polymerase chain reaction assay
Total RNA was extracted from the lung cancer cells and tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. cDNA was synthesized by random primers and the PrimeScript RT Reagent Kit (Takara, Dalian, China). The primer sequences for real-time PCR are shown in Supplementary Table 1. Real-time PCR was performed using SYBR Premix Ex Tag (Takara, Dalian, China). The PCR conditions were as follows: 95°C for 15 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. β-actin was used as the internal control.
Vector construction and RNA interference
The coding sequences (CDS) of human CSE1L was commercially synthesized (GENEWIZ), then was cloned and inserted into the lentiviral expression vector pWPXL. shRNA targeting CSE1L as well as a negative control (shNC) were obtained from GeneChem (Shanghai, China). To produce lentivirus containing CSE1L, 293T cells were cotransfected with the pWPXL- CSE1L and the lentiviral vector packaging system using Lipoectamine 2000.
Small-interfering RNA (siRNA) oligonucleotides for CSE1L and P65 were designed and synthesized by RiboBio (Guangzhou, China). The sequences for the siRNAs are shown in Supplementary Table 2. Transient transfection was performed using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. After transfection for 48 h, the cells were used for functional assays, including apoptosis, migration, invasion, colony formation, RNA extraction, and Western blot.
Protein extraction and Western blotting
Cell and tissue proteins were extracted using T-PER® Protein Extraction Reagent (Thermo Scientific, USA) with a phosphatase inhibitor cocktail (Roche Applied Science, Switzerland) and a proteinase inhibitor cocktail (Roche Applied Science, Switzerland). We used the MinuteTM Cytoplasmic and Nuclear Extraction Kit (Invent Biotechnologies, USA) to separate the nuclear and cytoplasmic. The protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). Proteins were separated by SDS-PAGE gels and transferred to nitrocellulose (NC) filter membranes (Millipore, Massachusetts, USA) or polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). The membranes were incubated with primary antibodies overnight at 4°C and probed with secondary antibodies at room temperature for 1~2 h. The antibodies used were shown in Supplementary Table 3.
Cell proliferation and colony formation assay
The relative cell proliferation was monitored using CCK-8 (Dojindo, Japan) according to the manufacturer’s protocol. Briefly, cells were seeded in 96-well plates at 1×103 cells per well. According to the manufacturer’s instructions, 10 μl of CCK-8 solution and 100ul medium was added to each well, and the mixture was incubated at 37°C for 2 h. The absorbance was measured at 450 nm.
For the colony formation assay, a total of 0.5 × 103 cells were seeded in 6-well plates and cultured at 37°C in a humidified incubator at 5% carbon dioxide. Two weeks later, the cell colonies were washed with PBS, fixed with methanol, and stained with 0.1% crystal violet (1 mg/mL) for 30 minutes. All of the experiments were repeated in triplicate and assessed under a light microscope.
Flow cytometry for cell apoptosis and cell cycle analysis
The human NSCLC cell lines (PC-9, H1299) stably interference CSE1L or the lentiviral vector or transfected with p65 si-RNAs or si-NC, A549 cells and H292 cells stably expressing CSE1L or the lentiviral vector and transfected with p65 si-RNAs or si-NC were used for the experiments. A flow cytometer (BD LSR II, BD Biosciences, USA) was used to detect the cell phenotype. Apoptosis was measured via the Apoptosis Detection Kit (BD Biosciences, USA) according to the manufacturer’s protocol. The data was analyzed using FlowJo software version 8.6.3 (FlowJo, USA). Cell cycle distribution was measured by the PI Cell Cycle Detection Kit (Beyotime Biotechnology, China) according to the manufacturer’s protocol. The results were analyzed using ModFit software (BD Biosciences, USA).
Cell migration and invasion assays
Cell migration and invasion assays were performed by Transwell filter chambers (BD Biosciences, New Jersey, USA). For migration assays, 5×104 cells in 200-µL of serum-free culture medium were suspended into the upper chamber with the noncoated membrane. For invasion assays, 1×105 cells in 200-µL of serum-free culture medium were placed into the upper chamber with the Matrigel-coated membrane diluted with serum-free culture medium. An 800-µL culture medium supplemented with 10% FBS was added in the lower chamber. After incubation at 37 °C in a humidified incubator under 5% carbon dioxide, the cells in the bottom surface of the membrane were fixed with 100% methanol, stained with 0.1% crystal violet for 30 min, and counted under a light microscope (Olympus, Japan).
Cells were plated in six-well plates on glass coverslips. The cells were fixed with 4% formaldehyde for 15 minutes at 40C, then infiltrated with 0.3% Triton X-100 for 15 minutes. After washing three times with PBS, the wells were treated with blocking solution for 30 minutes and then incubated overnight with anti-CSE1L (abcam, 1:50) and anti-P65(CST, 1:50) antibody at 4℃. After incubation with the secondary antibody (anti-rabbit (invitrgen, 1:300), anti-mouse (invitrgen, 1:150)) at room temperature for 1 hour, DAPI was used to stain the nucleus. A confocal laser scanning microscope (Leica TCS-SP5, Leica Microsystems, Germany) was used to observe the images.
Co-immunoprecipitation and mass spectrometry
When the cells fullness reached more than 90%, the cells were scraped off directly with a cell scraper using ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) with protease and protein phosphatase inhibitors (Roche Applied Science, Switzerland). Three milligrams of protein were pre-cleared with 30 μl of protein A/G magnetic beads (Millipore, Massachusetts, USA) at 4°C for 2 h. The beads were removed, and 5 μl of the primary antibody (CSE1L or P65) or isotype IgG was added to the supernatant at 4°C overnight with gentle mixing on a rocking platform to capture the fusion proteins. Then, 40 μl of protein A/G magnetic beads was added to each immunoprecipitation mixture for 4 h at 4°C. The magnetic beads were collected by placing the tube in the appropriate magnetic separator. The beads were washed three times with PBS/0.1% Triton. The magnetic beads isolated using a magnetic rack and then boiled in 2× sodium dodecyl sulfate (SDS) loading buffer. The bound fusion proteins were separated by SDS-PAGE and stained with a Silver Staining Kit (Beyotime Biotechnology, Shanghai, China). The proteins in the SDS-PAGE were digested with trypsin, and then analyzed by Triple TOF 5600 mass spectrometer (AB Sciex, Texas, USA). Protein identification was performed using Protein Pilot 4.5 (AB Sciex, Texas, USA).
In vivo tumor growth
All animal experiments were approved by the Animal Ethics Committee of the Shanghai Cancer Institute. Six- to eight-week-old female BALB/c-nu/nu mice were bred by Shanghai Cancer Institute (Shanghai, China) and housed in specific pathogen-free (SPF) conditions in a laboratory animal facility.
For the in vivo xenograft assays, 3×106 A549 cells and H292 cells stably expressing CSE1L or the lentiviral vector and 2×106 PC-9 cells and H1299 cells stably expressing shCSE1L or the negative control were separately subcutaneously inoculated into the dorsal right flanks of the nude mice (6 or 8 per group). The tumor size was measured two times every week. The tumor volume (V) was measured by calipers and calculated according to the following formula: (length × width × width)/2. After eight or ten weeks, the mice were sacrificed, and the tumors were weighing.
Differences among variables were assessed by χ2 analysis or two-tailed Student’s t-tests. Difference in survival were analyzed using the log-rank test. The correlation of CSE1L and P65 expression was examined by Spearman’s correlation test. Data are presented as the mean ± standard deviation (SD) or the mean ± standard error of the mean (SEM). Differences were considered statistically significant at P<0.05.