1. Cell culture, Lentivirus infection and transfection
The esophageal squamous carcinoma cell lines KYSE-150 and TE-1 were purchased from the Cell Bank of the Chinese Academy of Sciences Typical Culture Preservation Committee (Shanghai, China). Cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were cultured in a 5% CO2 incubator at 37°C. Radioresistant cells were generated from parental KYSE-150 and TE-1 cells by seeding the cells into 6-well plates and then exposing them to radiation at a dosage of 2 Gy per day for 30 days. The surviving cells were characterized as radioresistant cells (termed KYSE-150R and TE-1R cells). The generation of radioresistant cells was validated by colony formation assays.
sh-NORAD lentivirus, sh-EEPD1 and their corresponding control lentivirus were purchased from Genechem (Shanghai, China). miR-199a-5p mimics, miR-199a-5p inhibitors, siRNA against PUM1 and EEPD1-expressing vector were purchased from GenePharma (Shanghai, China). For virus infection, KYSE-150 and TE-1 cells were seeded into 48-well plates and grown to 50% confluence. Lentivirus (1×108 TU/ml) and infection reagents were mixed and added to the cells. The medium was changed after 48 hours of infection, and puromycin (3 µg/ml) was added to the medium to select cells that were successfully infected. Transient knockdown or overexpression of candidate genes was achieved by transfection of relative siRNAs or gene overexpression plasmid. Transient transfections were performed by using Lipofectamine 3000(Invitrogen/Thermo Fisher Scientific) according to standard protocol.
Total RNA was extracted from KYSE-150 and TE-1 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then reverse transcribed into cDNA using a PrimeScript™ RT reagent kit (TaKaRa, Dalian, China). MicroRNA-specific cDNAs were generated by using an EvoM-MLV RT kit. qRT-PCR was carried out using SYBR Premix Ex TaqTM II (TaKaTa, Dalian, China). GAPDH and U6 were used as reference genes for mRNA and microRNA, respectively.
3. Western blot
Total protein from KYSE-150 and TE-1 cells was extracted by RIPA buffer (Sigma Aldrich, Cambridge, MA) and quantified by BCA (Sigma Aldrich, Cambridge, MA). Then, proteins were subjected to SDS-PAGE through 10% gels and transferred onto PVDF membranes, which were then incubated with primary antibodies at 4℃ overnight. Secondary antibodies were then incubated with the membranes for 1 hour at room temperature before the protein bands were visualized using an ECL kit.
4. Extraction and identification of exosomes
Electron microscopy was used to observe and identify the extracellular vesicle-like structure of exosomes. Nanoparticle tracking analysis (NTA) was conducted to test the particle size-concentration distribution on a ZetaView PMX 110 (Particel Metrix, Germany). CD63 was considered an exosome marker, and protein expression was evaluated by western blot. For the exosome tracer experiment, 2000 cells/well were seeded in a 96-well plate; the exosome suspension was incubated with 2 µM PKH26 for 5 minutes at room temperature and added to the target cells in a volume of 20 µl/well. The red fluorescence indicates the process of exosomes entering target cells.
5. Fluorescence in situ hybridization
We used KYSE-150 and TE-1 to produce cell slides, which were pretreated with HCl and then fixed with neutral formalin. A probe targeting NORAD was used for hybridization with cell slides after treatment with diluted prehybridization solution. DAPI was used to stain nuclei, and cell slides were observed by confocal microscopy.
KYSE-150 and TE-1 cells were seeded on glass sides in 48-well plates and cultured overnight. Cells on the slides were fixed with 4% paraformaldehyde before they were treated with Triton X-100 (5%) to permeate cells and BSA solution to block nonspecific binding. Cells were treated with primary antibodies and incubated at 4°C overnight. After unbound primary antibody was removed, the cells were then incubated with PE-conjugated secondary antibody for 1 hour at 37 ℃. DAPI was added to the slides for nuclear staining, and glycerin was used to block the slides. Images of the slides under a confocal microscope were obtained. The primary antibodies and PE-conjugated secondary antibody were purchased from Abcam.
Cells were lysed at 4°C for 5 min in RIPA buffer, containing protease inhibitors. Whole cell lysates were then precleared with protein A/G beads. PD-L1 antibody was then added overnight at 4 ℃ . The antibody/antigen complex was pulled out of the lysates by using protein A/G-coupled agarose beads. Beads were washed by RIPA buffer for 3 times and resuspended by 2loading buffer. The Western blot and assays were described in previous.
8. RNA immunoprecipitation (RIP)
We purchased a Magna RIP™ RNA-binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) to analyze the interaction between RNA and proteins. KYSE-150 cells were lysed in RIP lysis buffer for further experiments. We used Ago2 antibody for immunoprecipitation and IgG antibody as the negative control. The expression levels of NORAD and pri-miR-199a were evaluated by qRT-PCR.
9. Homologous recombination (HR) reporter assay
HR reporter and I-SceI expression plasmids were purchased from Genechem (Shanghai, China). For the HR reporter assay, KYSE-150-sh-NC and KYSE-150-sh-NORAD cells were transfected with the HR reporter. G418 (1 mg/ml) was used to select successfully transfected cells. Then, these cells were transfected with the I-Scel plasmid to induce double-strand breaks on the HR reporter plasmid. Cells were harvested for analysis after 48 hours. Cells that underwent HR repair are positive for green fluorescence positive. Lentiviruses containing sh-NORAD and sh-NC used here were not fluorescently labeled.
10. Apoptosis and cell cycle analyses
We used an Annexin V-APC 7-AAD Apoptosis Detection Kit I (BD Pharmingen TM, New Jersey, USA) for the apoptosis assay. NORAD knockdown and NC cells were harvested and resuspended (5×105 cells per sample) before 5 μl of Annexin V-APC and 7-AAD were added to the suspensions and incubated for 15 minutes at room temperature in the dark. Flow cytometry was used to evaluate the luciferase intensity of Annexin V-APC and 7-AAD. A Cell Cycle Staining Kit (BD Pharmingen TM, New Jersey, USA) was used for evaluating the cell cycle distribution. Cells were fixed with 70% ethanol for 2 hours at 4°C. Target cells were treated with DNA staining solution and permeabilization solution in darkness for 15 minutes at room temperature. Flow cytometry was used to evaluate the luciferase intensity at 24 hours after treatment.
11. Replication fork recovery
Immunofluorescence assays of BrdU foci (BrdU in double-stranded DNA) were used to evaluate replication fork recovery after exposing ESCC cells to radiation [19, 20]. BrdU was incorporated into double-stranded DNA, and the immunofluorescence intensity of BrdU foci represented activation of replication fork recovery. Cells were exposed to 8 Gy of radiation before BrdU was added to the culture medium at a final concentration of 10 µM and incubated with the cells for 30 minutes. Cells with active replication forks were indicated by the cells with positive BrdU foci under a fluorescence microscope. Cells that did not receive radiation treatment were used as the control group.
12. Colony formation assay
Cells (500, 1000, 2000, 4000, or 8000 per well) were seeded into 6-well plates and cultured overnight in a 5% CO2 incubator before they were exposed to 0, 2, 4, 6, or 8 Gy of X-ray radiation. Colonies containing more than 50 cells were counted after 2 weeks of incubation. The survival curve after radiation was fitted based on the single-hit multitarget model: SF = 1 − (1 − e-D/D0)n.
13. Tandem mass tag proteomic-based quantitative proteome analysis
Cells successfully infected with lentivirus containing sh-NORAD or sh-NC were sonicated in lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail) to extract total protein. The protein sample was pretreated with dithiothreitol, alkylated with iodoacetamide and diluted with TEAB. Then, the protein mixture was incubated with trypsin, desalted on a Strata X C18 SPE column (Phenomenex) and vacuum-dried. A tandem mass tag (TMT) kit was used for further labeling. The peptides were thereafter evaluated by tandem mass spectrometry (MS/MS) on a Q ExactiveTM Plus (Thermo) coupled to the UHPLC system. The data-dependent procedure alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion and an automatic gain control (AGC) of 5E4. The fixed first mass was set as 100 m/z.
14. Xenograft mouse model and radiation treatment
ESCC cells suspended in phosphate-buffered saline were mixed with Matrigel and were injected into the right flanks of nude mice (KYSE-150 model, 5×106 cells) and C57BL/6 mice (AKR model, 1×106 cells). After the tumor volume reached 50-100 mm3 in the KYSE-150 model and 150 mm3 in the AKR model, local radiation (2 Gy/day for 4 consecutive days) was administered to the tumor site. Mice receiving concurrent treatment were also injected with anti-PD-1 antibody at 0, 7, and 14 days after the first radiation treatment. The tumor volume was measured with calipers and calculated using the following formula: tumor volume=0.5´ width2 × length.
15. Whole exome sequencing
Agilent V6 Exon + UTR region probe was used. Primitive paired-end reads were screened to generate clean reads. Then BWA was used to compare the clean reads to H19 and rearrange the clean reads according to Karyotype by Picard. Then Samtools and Picard were used for redundancy removal. After that, GATK was used to generate the SNPs/INDELs variation information, which was annotated by VEP. Tumor mutation burden (TMB) was then calculated (TMB= total somatic mutations/total length of target region).
16. Statistical analysis
GraphPad Prism 8.2.1 and R 3.3.1 were used for statistical analysis and data visualization. Student’s t test was performed to test differences between two groups, and one-way ANOVA was used to test differences among multiple groups. P＜0.05 was considered statistically significant.