CEP20 promotes invasion and metastasis of non-small cell lung … – Nature.com

Ethics approval statement

All the human non-small cell lung cancer samples were obtained from Zhejiang Cancer Hospital with informed consent. This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The Ethics Committee in Clinical Research (ECCR) of The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital) approved this retrospective study (IRB-2022-268). The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

All patients signed an informed consent approved by the institutional Review Board.

Quantitative real-timepolymerase chain reaction (qRT-PCR) was performed using 2Synergy Brands Inc (SYBR) Green (Servicebio) on the Roche LightCycler480 II RT-PCR Detection System. The relative CEP20 expression was quantified by RT-PCR, and Actin was used as an internal reference. All the reactions were triplicated and were calculated using the comparative threshold method (({2}^{{ - Delta Delta {text{C}}_{{text{t}}} }})).

Cell lysates or microtubule pellets were subjected to western blotting analysis with anti-CEP20, Actin, or GAPDH antibodies (Sigma, St Louis, MO, USA). The blots were probed with either Alexa Fluor 680 or IRDye 800-conjugated secondary antibodies and detected using the Odyssey system (LI-COR Biosciences, Lincoln, NE, USA). The uncropped immunoblot images are presented in Fig. S8.

A549 and H1299 cells were cultured in a complete DMEM (10% FBS included) medium with 5% CO2 at 37C. H226 and H520 were cultured in a complete RPMI 1640 (10% FBS included) medium with 5% CO2 at 37C. Cells were split at approximately 80% confluence by first aspiring the medium, followed by washing with preheated sterile 1PBS buffer thrice. Trypsin was given for 1min to induce cell detachment at 37C, then terminated by adding an appropriate volume of the medium. The cell mixture was transferred into a 15mL Falcon tube and dissociated to form a single-cell suspension by pipetting up and down. An appropriate volume of suspension was added to a new plate for continuous culture.

The cells were cultivated to the logarithmic growth phase and passaged the day before transfection. When cells reached 2030% confluence, Lipofectamine RNAiMAX and the siRNAs (CEP20 RNAi-1 5-ACCACTAATGTTTGTAGAATT-3 CEP20 RNAi-2 5-ATGGATGACCACCTAAGAATT-3) were diluted with DMEM (FBS free) according to the corresponding transfection system. Each dilution was incubated for 5min, mixed well, and incubated for another 20min at room temperature. After adding the mixture into corresponding groups, cells were cultured for 6h in a 5% CO2 incubator at 37C. Subsequently, the medium was replaced with complete DMEM containing 10% FBS. After 4872h of transfection, the cells were observed under a fluorescent microscope to evaluate their condition and transfection efficiency for further analysis.

A549 and H1299 cells were transfected and subsequently cultured for 48h. Then cells were evenly passage to 96-well plates with 2103 cells per well. Cultured for 24, 48, 72 and 96h, cells were added with 20 L/well MTT solution (5mg/ml, Sigma, St. Louis, MO, USA) and incubated for 4h at 37C. Then the medium was discarded and added 150 L of dimethyl sulphoxide (DMSO) (Sigma, St. Louis, MO, USA), the cell proliferation was analyzed by measuring the absorption at 490nm. Cell growth curves were depicted by Graphpad Prism 9 software.

Cells were plated on 12-well plates (200 cells per well). The cell culture medium was changed every 2days. After 2weeks, the cells were fixed with 4% paraformaldehyde for 15min, then washed 3 times by phosphate buffered solution (PBS), and dyed with crystal violet staining solution for 30min.

Cells were transfected and subsequently cultured until they reached 100% confluence. The cells were then starved overnight using a bare medium (DMEM or RPMI 1640 with no glucose or FBS). Mechanical scratching (wound) was performed manually with a pipette tip (10l), and the medium was replaced with DMEM or RPMI 1640 containing 1% FBS. Cells were imaged every 12h. It is important to note that the same area of the wound was imaged consistently across time points.

Cells were transfected following the protocol described above. After 48h of transfection, cells were starved overnight using a bare medium (DMEM or RPMI 1640 with no glucose or FBS). Subsequently, cells were trypsinized and counted, and 80,000 starved cells were resuspended in DMEM or RPMI 1640 containing 1% FBS and added to the upper chamber of Transwell inserts. The lower chamber was filled with 600l of complete DMEM or RPMI 1640 containing 10% FBS. The cells were then cultured for 4h at 37C with 5% CO2. After incubation, the transwell chambers were taken out and fixed with 4% PFA for 20min at room temperature. The inserts were stained with crystal violet for 20min, then washed for 5min each thrice. Residual non-migratory cells from the upper chamber were wiped off with a swab, while the migratory cells were counted and imaged under a microscope.

A549 cells grown on coverslips were fixed with cold methanol (20C), stained with anti-CEP20, -tubulin antibodies (Sigma, St Louis, MO, USA) for 2h at room temperature, and incubated with either Cy3-conjugated anti-mouse IgG or FITC-conjugated anti-rabbit IgG secondary antibody (Jackson ImmunoResearch) for 40min. DNA was stained with DAPI (Sigma). Finally, the mounted coverslips were analyzed by confocal fluorescence microscopy (LSM510, Zeiss).

For the cellular microtubule depolymerization assay21, A549 cells were treated with 5M nocodazole for the indicated times, and then centrifuged at 100 000g for 20min at 25C. For the cellular microtubule regrowth assay, A549 cells grown on coverslips were incubated with 5M nocodazole for 3h to depolymerize microtubules, and then carefully washed out to remove nocodazole followed by fixation at the indicated times. All cells were stained with mouse anti--tubulin primary antibody and Cy3-conjugated anti-mouse IgG secondary antibody. The coverslips were then mounted and imaged by confocal microscopy (NIKON, Tokyo, Japan). The astral length of microtubules within the region of interest were quantified using ImageJ software (Fiji, NIH). Data are expressed as means.d. and analyzed by students t-test. The supernatant and pellet fractions were collected separately and analyzed by western blotting with anti -tubulin.

Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturers instructions. The RNA amount and purity of each sample were quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed using Bioanalyzer 2100 (Agilent, CA, USA) with a RIN above 7.0 and confirmed by denaturing agarose gel electrophoresis. Poly (A) RNA was purified from 1g total RNA using Dynabeads Oligo (dT) 25-61005 (Thermo Fisher, CA, USA) with two rounds of purification. The purified poly(A) RNA was fragmented into small pieces using the Magnesium RNA Fragmentation Module (NEB, e6150, USA) at 94C for 57min. The cleaved RNA fragments were then reverse-transcribed using SuperScript II Reverse Transcriptase (Invitrogen, cat. 1896649, USA). The resultant cDNA was used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, m0209, USA), RNase H (NEB, m0297, USA), and dUTP solution (Thermo Fisher, R0133, USA). An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After treatment with the heat-labile UDG enzyme (NEB, m0280, USA) to remove the U-labeled second-stranded DNAs, the ligated products are amplified using PCR. The PCR conditions were as follows: initial denaturation at 95C for 3min; 8 cycles of denaturation at 98C for 15s, annealing at 60C for 15s, and extension at 72C for 30s; and final extension at 72C for 5min. The average insert size for the final cDNA library was 30050bp. Finally, the 2150bp paired-end sequencing (PE150) was performed on an Illumina Novaseq 6000 according to the manufacturers protocol.

Raw RNA-seq data were processed using fastp (v0.20.1)25 to remove adapter sequences and reads with low sequencing quality. The remaining clean reads were aligned to the human genome (hg38) using HISAT2 software (v2.1.0)26 with default parameter settings. Transcript assembly was performed using StringTie software (v2.0)27, and expression of transcripts sharing each gene_id was quantified as Transcripts Per Million (TPM). Differential expression analysis was performed using the R package DESeq228 with a threshold of significantly differentially expressed genes set as fold change (FC)>1.5 or<0.67 and adjusted P value<0.05. Heatmaps were generated using the R package pheatmap. The Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses in current study were done by R package clusterProfiler29. Adjusted p value<0.05 was considered as statistically significant. The gene set enrichment analysis (GSEA) was performed by R package enrichplot.

The dataset GSE1980430 based on the platform of GPL570 (Affymetrix Human Genome U133 Plus 2.0 Array) containing 30 paired gene-microarray samples of human NSCLC tumor and normal tissues were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo). The RNA-seq data of NSCLC samples were retrieved from the Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/), including 513 lung adenocarcinoma (LUAD) tumor samples, 57 LUAD adjacent normal samples and 501 lung squamous cell carcinoma (LUSC) tumor samples, and 49 LUSC adjacent normal samples. The expression levels of CEP20 were extracted from these datasets, and the NSCLC samples from the TCGA database were classified into three groups based on their CEP20 expression levels: relatively high (CEP20-high, n=253), relatively low (CEP20-low, n=253), and medium (CEP20-median, n=508).

All experiment results are presented as meanstandard deviation (SD) from 3 independent experiments and showed successful reproducibility. All graphs were generated using GraphPad Prism9 (64-bit, La Jolla, CA, USA). Two-tailed unpaired t-tests (Students t-test) were used to obtain the p values. The data are presented as the meanstandard deviation. *P<0.05, **P<0.01, ***P<0.001.

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CEP20 promotes invasion and metastasis of non-small cell lung ... - Nature.com