Long non-coding RNA stabilizes the Y-box-binding protein 1 and regulates the epidermal growth factor receptor to promote lung carcinogenesis

Indoor and outdoor air pollution has been classified as group I carcinogen in humans, but the underlying tumorigenesis remains unclear. Here, we screened for abnormal long noncoding RNAs (lncRNAs) in lung cancers from patients living in Xuanwei city which has the highest lung cancer incidence in China due to smoky coal combustion-generated air pollution. We reported that Xuanwei patients had much more dysregulated lncRNAs than patients from control regions where smoky coal was not used. The lncRNA CAR intergenic 10 (CAR10) was up-regulated in 39/62 (62.9%) of the Xuanwei patients, which was much higher than in patients from control regions (32/86, 37.2%; p=0.002). A multivariate regression analysis showed an association between CAR10 overexpression and air pollution, and a smoky coal combustion-generated carcinogen dibenz[a,h]anthracene up-regulated CAR10 by increasing transcription factor FoxF2 expression. CAR10 bound and stabilized transcription factor Y-box-binding protein 1 (YB-1), leading to up-regulation of the epidermal growth factor receptor (EGFR) and proliferation of lung cancer cells. Knockdown of CAR10 inhibited cell growth in vitro and tumor growth in vivo. These results demonstrate the role of lncRNAs in environmental lung carcinogenesis, and CAR10-YB-1 represents a potential therapeutic target.

time PCR was performed in the StepOne™ Real-Time PCR System (CFX96; Bio-Rad, Hercules, CA, USA) using SYBR® Premix Ex Taq TM (Takara Biotechnology). RT-PCR was performed with rTaq DNA polymerase (Takara Biotechnology) according to the manufacturer's instructions. The sequences of primers were listed in Table S6.

Microarray analysis
Tumor and paired normal lung tissues harvested from 10 NSCLCs were used for lncRNA microarray analysis. Briefly, 1 μg total RNA from each sample was amplified and transcribed into fluorescent cRNA along with the entire length of the transcripts without 3' bias utilizing a random priming method. The labeled cRNAs were hybridized onto the Human LncRNA Array v2.0 (8 x 60K, Arraystar, Rockville, MD, USA). After being washed, the arrays were scanned with the Agilent Scanner G2505B (Agilent Technologies, Santa Clara, CA, USA). Agilent Feature Extraction software (version 10.7.3.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies). The differentially expressed lncRNAs and mRNAs with statistical significance were identified using the volcano plot filtering. The threshold we used to screen dysregulated lncRNAs and mRNAs was fold change ≥ 2 and a p-value ≤ 0.05.

Vectors and transfection
The full-length CAR10 was amplified by PrimerSTAR Max DNA Polymerase (Takara Biotechnology) and sub-cloned into the NheI and Hind III sites of pcDNA3.1 (-) vector (Invitrogen) and pGEM-T easy vector (Promega). The cDNA sequence of EGFP with KOZARA motif was amplified from pEGFP-N1 vector and sub-cloned into the NheI and Hind III sites of pcDNA3.1 (-) vector. The cDNA sequence of EGFP was inserted into the ApaI site of pcDNA3.1-CAR10 plasmids without frame shift, and named pcDNA3.1-CAR10-EGFP. The full-length of CAR10 was inserted into the EcoR V and Hind III sites of pcDNA3.1-12MS plasmid to obtain the pcDNA3.1-12MS-CAR10 plasmid. The CAR10 deletion fragments were sub-cloned into the EcoR V site of the pCDNA3.1-12MS plasmid by One Step Cloning Kit (Vazyme TM , Nanjing, Jiangsu, China). The CDS sequence of YB-1 was cloned into the Hind III and BamH I sites of pcDNA3.1-Flag and pEGFP-N1 vector. Transient transfection of plasmids or siRNAs was conducted using the Lipofectamine 3000 kit (Invitrogen) according to the manufacturer's instructions. The double-stranded siRNAs and their negative control siRNAs (siNCs) (GenePharma, Shanghai, China) were transfected into cells at a final concentration of 50 nM. The cells were harvested at 24, 48 or 72 hour after transfection according to experimental needs.
The design of shRNAs was assisted by the use of a web-based software provided by Invitrogen (http:// rnaidesigner.invitrogen.com/rnaiexpress/), and the sequences were listed in Table S6. After annealing, double-strand oligos were inserted to the liner lentiviral vector pLKO.1, which was kindly provided by Prof. Wanzhu Jin at the Institute of Zoology, Chinese Academy of Sciences. pLKO.1 shRNA plasmid, psPAX2 packaging plasmid and pMD2.G envelope plasmid were co-transfected into the HEK293FT cells to produce lentivirus with control and CAR10 shRNAs. Infectious lentiviruses were harvested at 36 hours post transfection and filtered through 0.45 μm PVDF filters and named shNC, shCAR10-1 and shCAR10-2. A549-luciferase cells were infected with lentivirus in DMEM containing 8 μg/ ml polybrene (Sigma-Aldrich, St. Louis, MO, USA), and selected with 1 μg/ml puromycin (Amresco, Solon, OH, USA) for four weeks. The expression of CAR10 in the selected cells was tested by qRT-PCR.

Proteins and western blotting
The cells and tissues were lysed in RIPA buffer supplemented with protease inhibitors cocktail (Sigma). Subcellular fractionation proteins were extracted with nuclear and cytoplasmic Protein Extraction Kit (Beyotime, Haimen, Jiangsu, China) according to the manufacturer's instructions. The proteins were electrophoresed by SDS-PAGE under denaturing conditions and transferred onto the nitrocellulose membranes (Millipore Corporation, Darmstadt, Germany). Membranes were blocked in 5% non-fat milk (Thermo Fisher Scientific, Basingstoke, UK) and then incubated with primary antibodies. After being washed, the membranes were incubated with secondary antibodies, and detected by Luminescent Image Analyzer LSA 4000 (GE, Fairfield, CO, USA). Antibodies used in this study were: anti-YB-1, anti-EGFR, anti-AKT, anti

RNA pull-down assay
The full-length CAR10 was cloned into the pGEM-T easy Vector (Promega). Biotin-labeled CAR10-sense and CAR10-antisense were transcribed in vitro using a biotin labeling dNTP mix (Roche, Basel, Switzerland) and T7 polymerase (Promega) from linearized pGEM-T-CAR10 plasmid following the manufacturer's instructions. A549 cells (1.5×10 7 ) were collected for each group and washed with DEPC-PBS for three times. The cell pellets were re-suspended in 1 ml ice-cold cytoplasm isolation buffer (10 mM Hepes, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.05% NP-40 ) and vortexed at highest speed for 10 sec, followed by incubating on ice for 1 min. Nuclei were harvested at 1000 g for 3 min at 4°C. Pellets were re-suspended in 1 ml freshly prepared lysis buffer (50 mM Tris PH7.4 ; 150 mM NaCl, 1% NP-40, 0.5% Triton x-100, 10% glycerol, 1 mM PMSF, 1 mM cocktail, 200U/ ml RNaseOUT) and lysed with sustaining vortex for 30 min at 4°C . The nuclear lysates were obtained at 16,000g for 15 min at 4°C and pre-cleared with 20 μl pre-washed Streptavidin agarose beads (Invitrogen) at 4°C for at least 1 hour with gentle rotation. Biotinylated RNA (30 μg) was restructured in RNA structure buffer (10 mM Tris pH 7.0, 0.1 M KCl, 10 mM MgCl 2 ) at 90°C for 2 min, immediately transferred on ice and incubated for 2 min, then incubated at room temperature for 20 min to allow proper RNA secondary structure formation. Folded RNA was then mixed with 900 μL of nuclear lysate and tRNA (0.1 mg/ml) at 4°C for 6 hours. Prewashed streptavidinagarose beads (50 μl) were added into the pre-cleared nuclear lysate, and then incubated for 7 hours at 4°C. At the end of the incubation, the mixture was centrifuged at 1000 g at 4°C for 3 min and the supernatant was discarded. The beads were washed with ice-cold wash buffer (50 mM Tris pH7.4; 150 mM Nacl, 1% NP-40, 0.5% Triton x-100, 1 mM PMSF, 1 mM cocktail, 200 U/ml RNaseOUT) for seven times then boiled with 2× SDS loading buffer. The proteins were detected by Western blot.

RNA-immunoprecipitation (RIP)
For RIP, 1×10 7 A549 or H460 cells were collected and washed for 3 times with DEPC-PBS. Cells were resuspended in 1 ml ice-cold cytoplasm isolation buffer (10 mM Hepes, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.05% NP-40) and vortexed at highest speed for 10 sec, followed by incubating on ice for 1 min. Nuclei were harvested at 1000 g for 3 min at 4°C. Pellets were re-suspended in 1 ml freshly prepared RIP buffer (25 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5 mM DTT, 1% NP40, 0.5% Triton-100, 1 mM PMSF, 1 mM cocktail 100 U/ml RNaseOut), then lysed with sustaining rotation for 30 min at 4°C. The nuclear lysates were harvested by centrifugation at 16,000 g for 15 min at 4°C. Then 2.5 μg of anti-YB-1 antibody or control IgG (Santa Cruz) were added to the supernatant and incubated for 6 hour at 4°C with gentle rotation. 35 μl protein A/G sepharose beads (Santa Cruz) were added to each sample and incubated at 4°C for 6 hours. Beads were harvested at 1000g for 3 min. The beads were washed for seven times. The pellets were then re-suspended in 50 μl lysis buffer supplemented with 5 μl protease K and incubated at 55°C for 30 min, followed by addition of 1 ml TRIzol reagent (Invitrogen). RNA extraction was performed according to manufacturer's instruction, and the abundance of RNAs was detected by real-time RT-PCR.

Protein-RNA complex immunoprecipitation by 2MS2-12MS system
HEK293T cells were transfected with the 2MS2-12MS system plasmids. The cells (1×10 7 ) were used for each immunoprecipitation assay. 48 hours after transfection, cells were harvested and washed twice with ice-cold DEPC-PBS. Then the cell pellets were resuspended in 1 mL lysis buffer (20 mM Tris, pH 7.4, 10 mM NaCl, 2 mM EDTA, pH 8.0, 0.5% Triton-100, 1 mM PMSF, 1 mM proteinase inhibitor cocktail, 0.1 mg/ml tRNA, 200 U/ml RNaseOut) and lysed with sustaining rotation for 30 min at 4°C. Cell lysates were obtained by centrifugation at 16,000 g for 15 min at 4°C. 5% of cell lysates were used as input sample. Prewashed Flag-M2 beads (50 μl) were added into the lysates and incubated for 7 hours at 4°C. The beads were collected and washed seven times with wash buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Triton-100, 1 mM PMSF, 1 mM proteinase inhibitor cocktail, 0.1 mg/ml tRNA, 200 U/ ml RNaseOut). Half of the beads were boiled with 2× SDS loading buffer, and the rest beads were harvested for RNA extraction.

Nuclear and cytoplasmic RNA fractionations
Nuclear and cytoplasmic RNAs were isolated as previous description [2] . Briefly, 1×10 7 cells were harvested by trypsinization and washed twice with DEPC-PBS. Cell pellets were re-suspend in 200 μl lysis buffer (10 mM Tris HCl, pH8.0, 140 mM NaCl, 1.5 mM MgCl 2 , 0.5% NP40, 2 mM VRC) and incubated on ice for 5 min. Cell lysates were centrifuged at 12000g for 3 min, and the supernatant (cytoplasm fraction) was collected. The remaining pellets (enriched with nuclei) were washed with 160 μl lysis buffer twice and re-suspend in 100 μl lysis buffer. 1 ml TRIzol Reagent was added to the cytoplasm and nuclei fractions, respectively. RNA isolation and RT-PCR were performed as described above.

Colony formation assay
For soft agar assay, the bottom layer was prepared with a 0.6% agarose (Amresco) solution in DMEM with 10% fetal bovine serum in 35 mm plates, and the gel was placed for 30 min at room temperature. 0.5×10 3 A549-luciferace cells with stable expression of control and CAR10 shRNAs were added into 0.3% top agarose solution diluted with culture medium, then carefully placed on top of the bottom agarose. The plates were incubated at 37°C with 5% CO 2 for 2 weeks, and cell colonies were stained using 0.05% crystal violet (Amresco) and counted. For colony formation on plates, 0.25×10 3 A549-luciferace cells with stable expression of control and CAR10 shRNAs diluted in culture medium were plated into 6-well plates, and the plates were incubated at 37°C in 5% CO 2 air for 1 week, and culture medium was replaced twice. Finally, cell colonies were stained with 0.05% crystal violet (Amresco) and counted.

Animal studies
The animal studies were approved by the Institutional Review Board of Institute of Zoology, Chinese Academy of Sciences. All animal studies were conducted according to protocols approved by the Animal Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences. Six-week-old female SCID Beige mice were kept in specific pathogen-free (SPF) environment and used for the animal assays. A549luciferase cells stably expressing control and CAR10 shRANs were trypsinized and harvested in PBS, then a total volume of 0.1 ml PBS containing 1×10 6 cells were injected into the tail vein of the animals (n=15 for each group). Twenty-five days later, the mice were anesthetized by mixture of oxygen/isoflurane inhalation and positioned with legs fully extended, injected with 150 mg/kg body weight D-luciferin substrate (Gold Biotechnology, St. Louis, MO, USA), and the bioluminescence signals were detected by the IVIS Spectrum Imaging System (Caliper Life Sciences; Hopkinton, MA, USA). Survival of the mice was evaluated from the first day of cell transplantation until death or became moribund, at which time points the mice were sacrificed.