Two EBV-negative NPC cell lines (5-8F and HONE1) and 1 EBV-positive NPC cell line (C666-1) were kindly provided by Professor Musheng Zeng, Sun Yat-sen University Cancer Center of Guangzhou, China. One EBV-positive NPC cell line (HNE1-EBV) was kindly provided by Professor S.-W. Tsao, University of Hong Kong, China. HEK293T cells were obtained from Cancer Research Institute Southern Medical University of Guangzhou, China. All cell lines were cultured in RPMI-1640 (Invitrogen,Carlsbad, CA, USA) supplemented with 10 % newborn cow serum (NCS; Hyclone, Invitrogen). All cells were maintained at 37 °C with 5 % CO2.
Patients and specimens
A cohort containing 62 NPC specimens with TNM staging was collected for the association analysis of the expression of EBV-miR-BART8 with pathological and clinical data (Supplementary Table 1-2). All clinical samples used for immunohistochemistry (IHC) analysis of the expression of PAG1 were collected from the Nanfang Hospital of Southern Medical University, Guangzhou, China (Supplementary Table 3).The biopsy specimens of the validation set were obtained from a clinical trial conducted by our study group (Clinical Trials.gov no.NCT01171235). Tumor stage was scored according to the American Joint Committee on Cancer staging system (7th edition).
Establishment of radioresistant cells
C666-1 cells were exposed to 6 Gy X-rays once every 2 wk for a total of 5 times (cumulative dose: 30 Gy), yielding C666-1R cells. After the final exposure, cells were cultured under normal conditions for 4 wk.
Colony formation assay for radiosensitivity and irradiation
Colony formation assays were performed to assess the radiosensitivity of cells after IR. Suspensions containing 200, 400, 800, 1600, and 3200 cells were seeded into 6-well plates and exposed to 0, 2, 4, 6, or 8 Gy (2 Gy perfraction), respectively, using a 6 MV X-ray beam from an Elekta linear accelerator (Precise 1120; Elekta Instrument AB, Stockholm, Sweden) at a dose rate of 220 cGy/min. Cells were incubated for 14 d until colony appearance. Colonies were fixed for 15 min with carbinol (Huada, Guangdong, China) and stained for 30 min with 0.1 % Giemsa (AppliChem, Darmstadt, Germany). Colonies containing >50 cells were counted.
Transwell migration and Boyden chamber invasion assays
For the transwell migration assay, 105 cells in 100 mL serum-free RPMI-1640 media were triplicate seeded in each fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning, Shanghai, China). Consecutively, 600 mL of RPMI-1640 supplemented with 10 % NCS was added to the bottom chamber. Both C666-1 and C666-1R cells were incubated at 37 °C with 5 % CO2 for 8 h. Concomitantly, 5-8F, HONE1, and HNE1-EBV cells were incubated for 8, 12, and 8 h, respectively. Cells adhered on the lower surface were fixed with 100 % methanol (Huada) at 25 °C for 15 min and stained with hematoxylin (Macklin, Shanghai, China) for 15 min. Cell numbers in 6 predetermined fields in each replicate were counted under the microscope (NiKon ECLIPSE 80i system,NiKon, Shanghai, China ). All assays were independently repeated at least 3 times. Cell invasion assays were performed similar to the migration assay except that the transwell membrane was precoated with 24 mg/mL Matrigel (R&D Systems, Minn, USA).
Lentiviral construction and transduction
Lentiviral particles containing the GV209 expression vector encoding 282-nt of the pri-EBV-miR-BART8 precursor that produces both BART8-5p and BART8-3p (H1-miRNA-CMV-EGFP-BART8), as well as randomized flanking sequence control (H1-miRNA-CMV-EGFP-mock) were purchased from GeneChem (Shanghai, China) and transduced into NPC cells following the manufacturer’s instructions. Then, puromycin (Genechem, Shanghai, China) was added to the medium at a final concentration of 3 μg/mL for a 5 d selection.
Animal experiments were approved by the Ethical Committee for Animal Research of Southern Medical University (protocol number: NFYY-2018-76) and conducted based on the state guidelines from the Ministry of Science and Technology of China. All nude mice (4-6 wk old, male) were purchased from the Central Animal Facility of Southern Medical University. The in vivo metastasis model was established by tail vein injection. Briefly, 1 × 106 NPC cells were suspended in 100 μL serum-free 1640 medium and injected into the tail vein of nude mice (10 mice in each group). Accordingly, 6 wk later, whole bodies, and freshly dissected internal organs, including lungs and livers were collected for fluorescence imaging with the LT-9MACIMSYSPLUS whole-body imaging system (Encinitas, CA, USA). Organs were fixed in 4 % paraformaldehyde (Macklin) for 48 h and transferred to gradient ethanol (Huada). Then, organs were embedded in paraffin (Shitai, Jiangsu, China), sectioned using a Leica RM2235 microtome (Leica Biosystems, Weizler, Germany) and processed for histological examinations.
Paraffin sections prepared from patients were applied to IHC staining for the detection of the levels of PAG1 protein, using the indirect streptavidin-peroxidase method. All antibodies used for IHC are listed in Supplementary Table 4. The intensity of immunostaining was scored as weak (1), medium (2), and strong (3). The extent of staining, defined as the percentage of positive staining cells, was scored as 1 (≤25 %), 2 (26–50 %), 3 (51–75 %), and 4 (>75 %). An overall expression score, ranging from 0 to 7, was obtained by adding the score of the intensity and that of the extent of staining. The final staining score was given as low expression (overall score of 1–4), or high expression (overall score of 4-7).
Wound scratch assay
Cells (5 × 105) were seeded in a 6-well culture dish and grown to 90 % confluence. A single wound was made in the center of the cell monolayer and cell debris was removed by washing twice with PBS (Invitrogen). Complete medium was added and 5-8F or HONE1 cells were allowed to migrate into the clearing area for 24 or 12 h, respectively. Wound closure areas were visualized under an inverted microscope with a 100× magnification, and the migrated areas were counted.
Cells were cultured on coverslips overnight, fixed with 4 % formaldehyde in PBS for 15 min at 4 °C and then permeabilized with 0.5 % Triton-X-100 (Beyotime, Shanghai, China) in PBS for 30 min. Subsequently, cells were blocked for nonspecific binding with 5 % milk in TBS (Invitrogen) and Tween-20 (Santa Cruz Biotechnology, Delaware, CA, USA) (TBST) at 25 °C for 30 min, and then incubated with E-cadherin, vimentin, PAG1, or HA antibodies (Supplementary Table 4) at 4 °C overnight. Consecutively, cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:500, Proteintech, Rosemount, IL, USA) and Alexa Fluor 647 goat anti-mouse IgG (1:500, Proteintech) at 37 °C for 1 h. Coverslips were mounted on slides using anti-fade mounting medium with DAPI (Invitrogen). Accordingly, IF images were acquired on an OLYMPUS confocal micrograph system, and analyzed using the FV10-ASW1.7 viewer software (Olympus, Japan).
RNA oligos and cell transfection
The control mimic, miR-BART8-3p mimic, control inhibitor, and miR-BART8-3p inhibitor were synthesized by Integrated DNA Technology (GenePharma, Suzhou, China) (Supplementary Table 5). The culture medium was changed to fresh RPMI-1640 with 10 % FBS 24 h before transfection. The mimics and inhibitors were transfected to cells using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) at a final concentration of 50 nM. The medium was changed again to fresh medium 6 h after transfection.
Total RNA was extracted using the TRIzol reagent (Invitrogen), and complementary DNA (cDNA) was synthesized with the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Accordingly, qRT–PCR analysis was performed in triplicate using the SYBR Premix ExTaq (TaKaRa). The primers used for amplification of genes of interest are listed in Supplementary Table 6. Quantification of EBV-miR-BART8 was conducted with TaqMan microRNA assays (ABI, Shanghai, China). Mature miRNAs were reverse transcribed, and qRT–PCR was performed using the All-in-One miRNA qRT–PCR Detection Kit (GeneCopoeia, Guangdong, China) following the manufacturer’s protocol. RPU6B and β-actin were used for normalizing the expression of miRNA and mRNA, respectively. Fold changes were calculated using the 2-ΔΔCq method.
Plasmid preparation and cell transfection
The GV230 expression vector (http://www.genechem.com.cn) containing the whole coding sequence of PAG1, and HA-vimentin, as well as the GV170 control vector were purchased from GeneChem (Shanghai, China). Plasmid DNAs were purified using the TIANprep Mini Plasmid Kit (TIANGEN, Beijing, China) and transduced into NPC cells following the manufacturer’s instructions.
RNA-deep sequencing was performed and analyzed in Aksomics, Inc, Shanghai, China. In brief, mRNAs were isolated from DNase-treated total RNA using the Dynabeads mRNA Purification Kit (Invitrogen) . According to the manufacturer’s instructions, mRNAs were fragmented with divalent cations and converted to single-stranded cDNA using random hexamer primers and Superscript II reverse transcriptase (Invitrogen). The second strand of cDNA was generated by RNase H (Enzymatics,Beverly, MA,USA ) and DNA polymerase (Enzymatics). Subsequently, cDNA products were purified using Ampure beads XP (Beckman, Indianapolis, IN, USA). After converting the overhangs into blunt ends using the T4 and Klenow DNA polymerases (Enzymatics), an extra "A" base was added to the 3′-end of cDNA by the Klenow enzyme. Sequencing adapters were then ligated to the end of cDNA by T4 DNA Ligase (Enzymatics). Fragments of 200 bp were selected using Ampure beads XP (Beckman) and enriched through 12 cycles of PCR. PCR products were loaded into a flowcell to generate clusters and then sequenced using the Hiseq 2000 system (Illumina). Selected results are shown in Supplementary Table 7.
MiRNA target predictions
EBV-miR-BART8 candidate targets were initially obtained from the RNA sequencing results (Supplementary Table 7) and then enriched through literature retrieval (Supplementary Table 8). The RNAhybrid program (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission. html) was used to predict the duplex complementation between the human PAG1 3′-UTR and miR-BART8-3p.
Luciferase reporter assays
The MiTarget microRNA 3′-UTR target vector (pEZX-MT01, Gene) containing the full-length 3′-UTR of PAG1 with binding sites for EBV-miR-BART8-3p (wild-type 3′-UTR) was provided by GeneCopoeia. The mutant site in PAG1 3′-UTR was then generated by site-directed mutagenesis using the KOD-Plus-Mutagenesis Kit (SMK-101, Toyobo C. Ltd, Life Science Department, OSAKA, JAPAN). For luciferase reporter assays, the wild type (wt) or mutant (mut) 3′-UTR vector was cotransfected with EBV-miR-BART8-3p mimic or nonspecific mimic control (Genepharma) into HEK 293T cells, respectively. Luciferase activity was measured 48 h after transfection using the Luc-Pair miR Luciferase Assay Kit (GeneCopoeia) on a Panomics Luminometer (Panomics Inc. Fremont, CA, USA).
Co-immunoprecipitation and mass spectrometry assays
To determine potential PAG1-binding proteins, HONE1 and 5-8F cells transfected with the empty vector, as well as flag-PAG1-expressing HONE1 and 5-8F cells were used for coIP employing anti-flag beads. The PAG1 protein complex was eluted using a 0.1 M blycine solution (Fude), separated on an SDS gel, visualized by silver staining using the silver staining kit (Invitrogen), and analyzed by MS at Genepharma (Supplementary Table 9). CoIP assays were performed using 1 mg cell lysates in NP-40 buffer (Fude), with anti-HA and anti-Flag antibodies being employed to pull down the PAG1 and vimentin protein, respectively.
This study was reviewed and approved by the Ethics Committee of Nanfang Hospital, Southern Medical University (Guangzhou, Guangdong, China) and was conducted in accordance with the Declaration of Helsinki.
All experiments were performed in triplicate. Data shown are mean ± s.e.m. (unless otherwise specified), from at least 3 independent experiments. The SPSS 16.0 software (IBM SPSS Statistics, Chicago, IL, USA ) was used for statistical analyses. Differences were considered to be statistically significant at values of P < 0.05 by Student’s t-test or χ2 test (categorical variables) for 2 groups, or by one-way ANOVA (analysis of variance) analysis for multiple groups. Correlations were analyzed using the two-tailed Spearman’s correlation analysis. Single, double, and triple asterisks indicate statistical significance *P < 0.05, **P < 0.01, and ***P < 0.001.