Urban particulate matter samples SRM 1648a (encoded as PM2.5) were purchased from National Institute of Standards and Technology (NIST, MD, USA). The PM2.5 was collected in an urban area and all constituents in SRM 1648a were naturally present in the material before processing (NIST Certificate of Analysis, 2020). SRM 1648a includes 25 metals, 21 polycyclic aromatic hydrocarbons (PAH), and 7 polychlorinated biphenyls (PCBs). As SRM 1648a has uniform constituents，it is usually used as a reference to estimate the impact of mixed PM2.5 on health damage and may also be used as the reference material . Before each test, PM2.5 was dissolved in phosphate buffer saline (PBS) for infusion and the samples were sonicated for 20 min before use in each experiment.
We determined the PM2.5 dosage for in vitro experiments on the basis of previous reports in PM-2.5-induced cytotoxicity and on our pilot cell viability experiments. BEAS-2B cells (the normal human bronchial epithelial cell line) were exposed to different levels of PM2.5 for 24 h and the cell viability was tested by CCK-8 methods. No significant changes of cell viability were observed between cells exposed to PM2.5 (25, 50, 100, 200 μg/mL, respectively) and control cells. When the concentration of PM2.5 reached 400 μg/mL, the cell viability significantly (P < 0.001) decreased to approximately 50% (≈LC50) (Fig. S1). This effect dose was similar to reports in which the dose of SRM 1648a to reduce cell viability to 50% was about 500 μg/mL . Considering that previous studies have also shown that the optimum doses of PM2.5 to induce apoptosis, inflammatory cytokine release, or DNA damage in cells were between 200 to 500 μg/mL [22,23,24,25,26]. we chose 100 μg/mL, 200 μg/mL, and 400 μg/mL as the working doses of PM2.5 for in vitro experiments in the present study.
The doses of PM2.5 used for animal experiment were estimated according to the World Health Organization (WHO) air quality guidelines (WHO, 2006) and physiological parameters of rats: The respiratory volume of an adult rat (200 g) is 0.86 mL at each breath and the breath rate is 85 times per min, and breathing quantity per day is 0.105 m3. According to the WHO annual mean concentration (target-1, 35 μg/m3), PM2.5 exposure level was calculated as 3.684 μg/per day. After multiplying by a 100-fold uncertainty factor, the exposure level of PM2.5 was estimated to be 1.8 mg/kg body weight (bw)/per day. Using 1.8 mg/kg bw as the low-dose, a 3-fold (5.4 mg/kg bw) and a 9-fold (16.2 mg/kg bw) doses were considered as moderate- and high-dose, respectively [27, 28].
This study was approved by the Medical Ethics Committee of Shenzhen University Health Science Center (Approved no. 2016002). Written informed consents were obtained from all participants. NSCLC subjects were recruited from patients who underwent surgical resection in the Department of Thoracic Surgery at People’s Hospital of Shenzhen, China (Supplementary Table S9). NSCLC was diagnosed according to the criteria of Lung Cancer Stage Classification (The Eighth Edition) . Tissue samples were collected in the operating room and the surgical specimens were fixed in formalin and embedded in paraffin before they were archived.
Immunohistochemistry (IHC) analysis
The paraffin-embedded tissue slides were deparaffinized and dehydrated in xylene and alcohol, and then incubated with the primary antibodies against DLAT (Sigma, R38114) at 4 °C overnight and then with HPR-labelled secondary antibody (MaxVision TM HRP, 210908S407n) at 37 °C for 1 h. The 3,3-diaminobenzidine (DAB) solution was used for development of brown staining to identify the expression of DLAT. Images were observed and analyzed under microscope (Olympus) with Image J software.
Cell culture and transfection
BEAS-2B and human NSCLC cell lines (A549, and PC9) were ordered from Shanghai Institute for Biological Science (Shanghai, China). Each cell lines were authenticated by measuring the short-tandem repeat (STR) DNA profiles. No contamination of mycoplasma was found in these cell lines. A549, PC9, and BEAS-2B cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, NY, USA) in a humidified atmosphere with 5% CO2 at 37 °C. For in vitro PM2.5 exposure experiments, cells were treated with different concentrations of PM2.5 (0, 200, 400 μg/ml) for 24–48 h.
Ribosome sequencing (Ribo-seq)
A total of 1×107 BEAS-2B cells were lysed in polysome lysis buffer (20 mM Tris•Cl at pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, Turbo DNase I 25 U/ml, 50 mg/mL cycloheximide). Then, cell lysate was treated with unspecific endoribonuclease RNase to digest the free RNAs other than ribosome-protected fragments (RPFs). Isolation of ribosome was performed by size-exclusion chromatography with MicroSpin S-400 HR columns (GE Healthcare, USA). Recovered RPFs were then subjected to rRNA depletion kit to remove rRNA contamination. Small RNA (< 200 bp) was resolved by electrophoresis on a 15% (w/v) polyacrylamide gel (PAGE) with 7 M urea. The RPFs bands (~ 30 nt) were cut for RNA extraction, phosphorylated and ligated with 5′ and 3′ adapters, reverse transcribed and PCR amplified, respectively. Libraries were then sequenced on Illumina Novaseq platform using the PE150 (paired-end 150 nt) recipe. The sequencing reads were mapped to human genome database by TopHat2 software and then quantified by the HTSeq and DESeq2 R packages.
BEAS-2B cells from the same cultures used in the Ribo-seq experiments were processed in parallel for RNA-seq. Total RNA was isolated with TRIzol reagent and RNA-seq libraries were prepared using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Briefly, mRNA was purified from total RNA using the poly-T+ oligo-attached magnetic beads. RNA samples were fragmented and then used for first- and second-strand cDNA synthesis with random hexamer primers. The cDNA fragments were treated with DNA end repair kit and NEBNext adaptor was ligated at the 3′ end of the DNA fragments. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA) and then subjected to PCR amplification. After cluster generation by the TruSeq PE Cluster Kit v3-cBot-HS (Illumia, USA), the library preparations were sequenced on an Illumina Novaseq platform (150 bp paired-end). Sequencing reads from RNA-seq were aligned to the reference genome (GRCh38) using Hisat2 v2.0.5. Feature Counts v1.5.0-p3 was used to count the reads numbers and gene expression levels were calculated by the Reads per kilobase of transcript, per millions mapped reads (RPKM). Differential expression analysis was performed using the DESeq2 R package (1.16.1).
The abundance of ribosome footprints of an mRNA depends on the translation rate and on the level of mRNA expression. Therefore, TE (translation efficiency) is commonly estimated as the ratio between RPF and expressed mRNA counts (mRNA) (TE = RPKM in Ribo-seq/RPKM in RNA-seq). Differences in TE between PM2.5-exposed and control samples were calculated using the Ribodiff software.
Animals and PM2.5 intratracheal instillation
4-week old male SD rats were purchased from Guangdong Experimental Animal Center (Foshan, China) and were housed in a pathogen-free environment with free access to food and water. After one-week adaption, rats were randomly divided into 4 groups (n = 5 per group): three experimental groups exposed to PM2.5 (1.8, 5.4, 16.2 mg/kg body weight)  by intratracheal instillation every 3 days for 24 days (in total 8 times), and one control group received equal volume of saline (Fig. S5A). At the end of experiments, the rats were euthanized, and the lung tissue were isolated and stored at -80 °C for subsequent analyses. All animal experiments were performed in accordance with the protocols approved by the Animal Ethical Committee of Shenzhen University.
Immunohistochemical (IHC) staining of rat lung tissues
The lung tissue was fixed in 4% paraformaldehyde solution. After paraffin embedding, 4 μm sections were cut and stained with hematoxylin and eosin to reveal the pathology characteristics of lung tissue. The slides were treated with graded alcohols for rehydration, and with EDTA buffer for 10 min at 98 °C for antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution at room temperature for 10 minutes. Nonspecific binding was prevented by blocking with normal goat serum at room temperature for 30 minutes. Sections of lung tissue were incubated with primary antibodies against Ki-67 and Caspase3 (Proteintech, Wuhan, China) at 4 °C overnight. Tissue sections were then treated with secondary antibody (goat anti-rabbit IgG H&L, HRP), stopped with freshly configured DAB solution, and counter-stained with hemetoxylin. The areas of positive staining were evaluated by ImageJ 1.53a (National Institutes of Health, USA).
Quantification of lactate and pyruvate
For in vitro measurement of L-lactate and pyruvate levels, 1 × 106 cells were seeded into each well of the six-well plate. Cells were treated with PM2.5, PBS, DLAT-overexpression or NC vector, respectively. For tissue analysis, 100 mg tissues were individually ground with liquid nitrogen, and the homogenate was resuspended with 0.4 ml 0.86% saline water and thoroughly vortexed. The samples were then centrifuged at 3500 rpm/min at 4 °C for 15 min. Then the supernatants were collected for further measurements. L-lactate and pyruvate levels in the cell cultured medium or in the supernatants of lung tissue homogenates were quantified using the L-lactate assay kit (A019–2-1, Jiancheng Bioengineering Institute, Nanjing, China) and the pyruvate assay kit (A081–1-1, Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instruction. The sample absorbance (530 nm for L-lactate and 505 nm for pyruvate) was detected using the Synergy HTX Multi-Mode Microplate Reader (BioTek, Guangzhou, China). All in vitro experiments were performed in triplicate.
Measurement of ECAR and OCR
The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of cells were assessed using the Seahorse XFe24 Flux Analyzer (Seahorse Bioscience, Agilent). The glycolytic stress test kit (Seahorse Cat. #103020–100) and mitochondrial stress test kit (Seahorse Cat. #103015–100) were used for ECAR and OCR detection, respectively. Briefly, 1 × 104 cells were seeded in the 96-well XF Seahorse incubation plate as the protocol indicated. Cells were cultured at 37 °C in XF base medium (pH 7.4), and glucose (10 mM), glutamine (1 mM), 2-DG (50 mM) and oligomycin (1 μM) were added sequentially into the plates at specific time points following the manufacturer’s guidelines. Data of OCR and ECAR were measured and plotted using the Seahorse XF24 software.
Plasmid construction, siRNA, and cell transfection
DLAT overexpression plasmid and the empty negative control (NC) were constructed using the pcDNA3.1-GFP vectors, respectively. siRNAs targeting DLAT, eIF4E, and Sp1, and corresponding NCsequences were designed and synthesized by RiboBio Co., Ltd. (Guangzhou, China). Cells were transfected with plasmids, siRNAs, or NCs, respectively, using the Lipofectamine 2000 reagents (Invitrogen, Shanghai, China) according to the manufacturer’s protocol. All sequences of siRNAs are listed in Supplementary Table S10.
Cell viability assay
Cell proliferation rate was determined with Cell Counting Kit-8 kit (Dojindo Molecular Technologies, Osaka, Japan) according to the manufacturer’s instructions. Briefly, cells were seeded into 96-well plates (3000 cells/well) and transfected with plasmid, siRNAs, or their corresponding NCs. After incubation for 48 h, 10 μl CCK-8 reagent was added to each well and then incubated for another 1 h. The absorbance at 450 nm was measured by a Synergy HTX Multi-Mode Microplate Reader (BioTek, Guangzhou, China). Each independent experiment was performed in triplicate.
Cell invasion was evaluated by Matrigel-coated Transwell chambers (Corning-Costar, NY, USA). Briefly, cells were incubated in the upper chamber of a 24-well Transwell plate. After transfection for 48 h, 5 × 104 cells were seeded into the upper compartment, which contained RPMI1640 medium with 10% FBS. After 24 h incubation, non-invading cells were gently removed and the migrated cells were fixed with methanol followed by DAPI staining. The number of invaded cells was counted under light microscope (Nikon, ECLIPSE Ts2, Japan).
Flow cytometric analysis was performed to analyze the percentage of apoptotic cells using the Annexin V-FITC cell apoptosis kits (BD Biosciences, CA, USA) according to the manufacturer’s protocol. In brief, cells were transfected with plasmids or NCs for 48 h, and then stained with Annexin V-fluorescein isothiocyante (FITC) and propidium iodide (PI). The apoptotic rates of cells were determined by flow cytometer (Beckman Coulter, Inc., IN, USA) equipped with CytExpert software (version 2.3).
Total proteins were extracted from cells or tissues using RIPA lysis buffer (Promega, Madison, WI, USA). Proteins were separated on an 8% SDS-PAGE gel and then transferred to PVDF membranes. The membrane was blocked with 5% skim milk at room temperature for 1 h and then incubated with primary antibodies (anti-DLAT, 1:1000 dilution, Abcam, ab51608; anti-SP1, ab101562, 1:1000 dilution, Abcam) at 4 °C overnight, followed by incubating with horseradish peroxidase (HRP) conjugated β-actin secondary antibodies (1:5000) at room temperature for 90 min. The protein bands were scanned and visualized by the GS700 imaging densitometer (Bio-Rad Laboratories) and analyzed by Image Studio software.
Total RNA was isolated using the TRIzol reagents (Life Technologies, Shanghai, China) according to the manufacturer’s instructions. 1 μg of total RNA was reverse-transcribed into cDNA using the Yeasen cDNA kit (11121ES60, Shanghai, China). The transcript level of specific gene was amplified using the Yeasen qPCR kit (11201ES08) and was normalized to ACTB. The primers were synthesized by Riobio Co. (Guangzhou, China) and the sequences are listed in the Supplementary Table S10.
Human lung tissue microarray
A tissue microarray containing slides of 92 NSCLC tumor tissues and adjacent normal lung tissue was purchased from Outdo Biotech, (Shanghai, China; HLugA180Su04). The patients’ clinical records and histopathological diagnoses were fully documented. Survival times was calculated in months and defined as the time from operation time until death, or censored if no death was noted at follow-up date. The protein level of DLAT was determined by semi-quantitative IHC assay, using the anti-DLAT antibody (Sigma-Aldrich，HPA040786). The results of IHC were independently scored by two independent observers. The positive stained samples were scored as follows: 1, ≤25% of positively stained cells; 2, > 25- ≤ 50% of positively stained cells; 3, > 50- ≤ 75% of positively stained cells; 4, ≥75% of positively stained cells. The intensity of staining was scored according to the following criteria: 0, negative staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The final staining index (SI) was calculated by multiplying the percentage score by the staining intensity score, with SI values ≥3 being defined as high expression and SI < 3 being considered as low expression.
Polysomal mRNA isolation and qRT-PCR
Isolation of ribosome-bound mRNA by polysome separation was performed as described previously with minor modifications. Briefly, A549 cells (1 × 107) were incubated in DMEM medium containing 100 μg/mLcycloheximide for 10 min at 37 °C. All subsequent steps contained 100 μg/mL cycloheximide. After centrifugation, resuspended cells were treated with trypsin at 37 °C for 3 min, then lysed by polysome extraction buffer (PEB; 20 mM Tris-HCl, pH 7.5, 50 mM KCl; 10 mM MgCl2; 1 mM DTT; 100 μg/ml CHX; 200 μg/ml Heparin) containing 1% Triton-X100. After 30 min incubation on ice, lysates were centrifuged at 14,000 rpm for 30 min at 4 °C, and the resulting supernatant were collected and then loaded onto 5–50% sucrose density gradients. Gradients were then centrifuged at 38000 rpm for 120 min in an Optima L-100XP rotor (Beckman Coulter) using equal OD260 units of samples. Thirteen fractions of equal volume were collected, and their absorbances were measured. For technical reasons, fractions 1–3, 4–6, 7–9, 10–11, and 12–13 were pooled together as 1, 2, 3, 4, 5 combined fractions, respectively . Messenger RNAs of 1 or 2 pooled fractions were light weight fraction (fractions 1–6) and mRNAs from 3, 4, 5 pooled fractions were heavy weight fraction (fractions 7–13). RNA was extracted from equal volume of the five combined fractions using TRIzol reagents (Life Technologies, Shanghai, China), reversely transcribed to cDNA using the PrimeScriptTM RT reagent Kit (Takara, Beijing, China) and amplified by qTR-PCR analysis using the TB Green Premix Ex TaqTM II kit (Takara, Beijing, China).
Chromatin immunoprecipitation (ChIP) assay
The Sp1-binding sites surrounding DLAT promoter region were predicted using the JASPAR tools (http://jaspar.genereg.net/). Primers were designed to amplify the predicted binding sequences of DLAT promoter regions (Supplementary Table S10). In brief, the A549 cells were crosslinked with 1% formaldehyde (final concentration) and lysed with ChIP Lysis Buffer at 37 °C for 10 min. Chromatin was sonicated into 200 ~ 1000 bp fragments. The lysates were then incubated and precipitated with antibodies against the Sp1 antibody (mAb #9389) or control IgG (CST，9389S), after which DNA-protein immunocomplexes were collected, using the PierceTM protein A/G-agarose beads (Thermo Fisher Scientific, Shanghai, China), and treated with RNase A (Sigma) and proteinase K (Sigma). The precipitated chromatin DNA was recovered and analyzed by qRT-PCR.
Dual luciferase reporter assay
To test the transcriptional influence of Sp1 on the DLAT promoter, wild-type and mutant DLAT promoter fragments containing the Sp1 binding site were cloned into the dual-luciferase reporter pGL3-basic, respectively. Sp1 was cloned into the pcDNA3.1 vector using the restriction enzymes EcoRI and XhoI. HEK293T cells were cultured at a density of 2 × 104 cells/well in 96-well culture plates and were incubated overnight. On the next day, the cells were co-transfected with pGL3-basic-DLAT-WT and Sp1-pcDNA3.1, pGL3-basic-DLAT-MUT and Sp1-pcDNA3.1, or corresponding control vectors, and the internal control vector pRL-TK (Promega, Madison, WI), respectively, using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA) according to instruction of the manufacturer. After 48 h post-transfection, luciferase activity was measured using the Dual Luciferase Reporter Gene Assay (Beyotime, Shanghai, China) on a Chemiluminescence analyzer (ZS/BK-L96C, Beijing, China). All vector constructs were verified by DNA sequencing. The primer sequences for the constructs are listed in Supplementary Table S10. At least three independent transfection experiments were carried out for each condition.
In silico analyses
The gene sequences of DLAT (NM_001372031.1), FOXM1 (NM_001243088.2), GAPDH (NM_001256799.3), β-actin (NM_001101.5), MYC (NM_001354870.1), Cyclin D1 (NM_053056.3) were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/gene/). The minimum free energies of 5′-UTRs of these genes and their corresponding secondary structures were predicted in silico using the online RNAfold software (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The associations of overall survival (OS) and relapse free survival (RFS) with the expression levels of eIF4E, Sp1, and DLAT mRNA in NSCLC patients were analyzed by the online tool KM Plotter (https://kmplot.com), using median as the cutoff values in the TCGA dataset. Gene and protein expression levels of eIF4E, Sp1, and DLAT in tumor and normal tissues were analyzed using the TCGA dataset with the online analyzers (http://gepia.cancer-pku.cn/; http://ualcan.path.uab.edu/).
Data are expressed as the means ± SD from at least three independent experiments. Statistical analyses were performed using SAS 9.4 software (SAS Institute, USA) and Graph Pad Prism 6 (Graph Pad, USA). Comparison between groups was conducted using Student’s t-test (for parametric data) or the Mann–Whitney test (for non-parametric data). The associations between DLAT expression levels and overall survivals of NSCLC patients were estimated by Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate Cox proportional hazards models were performed to identify the factors that had a significant effect on survival outcome. P-value (two-sided) < 0.05 was considered statistically significant.