3.1. Patients and specimens
All clinical samples were collected from the Department of Radiation Oncology at the First Affiliated Hospital of Nanjing Medical University. All subjects gave informed consent before they participated in the study. The study was conducted in accordance with the guidelines of the Declaration of Helsinki, and the protocol was approved by the Ethics Committee and Institutional Review Board of the First Affiliated Hospital of Nanjing Medical University. A total of 88 OSCC patients were recruited in this study between 2017 and 2019. Clinical staging was based on the seventh edition of the AJCC Cancer Staging Manual(13). All patients received a 60 Gy total dose of involved-field radiation (2 Gy per fraction, 5 days per week) plus two cycles of concurrent chemotherapy with cisplatin and fluorouracil and two cycles of post radiation chemotherapy. Radiation was delivered as described previously(14). Blood samples were collected one day prior to definitive chemoradiation. Follow-up studies included computed tomography, barium swallow, endoscopy and physical examination. All patients were followed up every 3 months until death or May 2020. In-field recurrence included primary lesions and involved regional lymph nodes inside the PTV. Overall survival was defined as the interval between completion of radiation and death(14). We collected 30 ml of whole blood from each patient into BD Vacutainer K2 EDTA Blood Collection Tubes (BD, USA), and we immediately centrifuged the whole blood at 4000× g for 10 minutes at 4 ℃ to obtain plasma. The plasma was then ultracentrifuged to collect EVs.
3.2. Cell culture and hypoxia treatment
Human OSCC cell lines (Te13, Te1 and Eca109) were obtained from the American Type Culture Collection (ATCC, USA). All cell lines were cultured in RPMI-1640 medium (Gibco, USA) with 10% foetal bovine serum (FBS; Gibco, USA), 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained at 37°C in 5% CO2 and were routinely examined for Mycoplasma contamination. To induce hypoxia (<1% O2), cells were cultured at 37°C in the same incubator in an AnaeroPack jar with AnaeroPack-Anaero (Mitsubishi, Japan) according to the manufacturer's instructions. The hypoxic environment was confirmed by detection of hypoxia inducible factor 1 subunit alpha (HIF-1α) expression. Cells were irradiated by RS 2000 Pro X-Ray Bio-irradiator (Radsource, USA) with 140kV X-ray beam. The irradiation field was confined within the culture dish or disk, and the dosing rate was 1.439Gy/min.
3.3. EV isolation and identification
FBS was depleted of EVs by ultracentrifugation at 140,000× g and 4°C for 16 hours, and the supernatant was collected and filtered through a 0.22 μm filter (Millipore, USA). EVs derived from blood samples and cell culture medium were isolated by differential centrifugation as previously described(15). Before EV isolation, cells were cultured in normal medium to 50% confluency and were then washed with phosphate-buffered saline (PBS) three times; the medium was then replaced with RPMI-1640 containing 10% EV-depleted FBS and cultured under normoxic or hypoxic conditions. After 48 hours, the cell culture medium was harvested (50 ml), and EVs were isolated by differential centrifugation as previously described. The EVs were used immediately for further experiments. The size distribution and concentration of EVs were analyzed by nanoparticle tracking analysis (NTA) using a ZetaView particle tracker from ParticleMetrix (Meerbusch, Germany). We used transmission electron microscopy (TEM; JEM-1200EX, JEOL Ltd., Japan) to observe the structure of EVs. CD63, CD81 and Alix were used as exosomal markers, and calnexin was used as the negative control marker for EVs. PKH67 (Sigma-Aldrich, USA) was used to label EVs. Twenty-four hours after PKH67-labelled EVs were incubated with OSCC cells, DAPI was used for nuclear staining. Cells were visualized with a confocal fluorescence microscope (Leica, Germany).
3.4. Western blotting
Samples of cells and EVs were washed and resuspended in RIPA lysis buffer (Beyotime, Shanghai, China) with a protease and phosphatase inhibitor mixture (Millipore). Proteins were separated based on their molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk powder in Tris-buffered saline containing Tween 20 (TBST) for 2 hours, and were then incubated at 4°C overnight with specific primary antibodies (details are listed in Table S1). The membranes were rinsed in TBST three times (10 minutes each time), incubated with secondary antibodies at room temperature for 2 hours and then washed again in TBST (three times, 10 minutes each time). Protein expression levels were measured with ECL Plus reagent (Millipore) using a Bio-Imaging System (Bio-Rad, USA).
3.5. Quantitative real-time PCR (qRT-PCR)
Following the manufacturer's instructions, total RNA was extracted with TRIzol reagent (Invitrogen, USA). For qRT-PCR of mRNA, total RNA was further reverse transcribed into cDNA using a PrimeScript RT Reagent Kit (Takara, Japan). For RT-PCR of miRNA, we used a Mir-X miRNA First-Strand Synthesis Kit (Takara) to perform reverse transcription. The reactions were performed in a real-time PCR system (Applied Biosystems 7500, USA) with a TB Green Premix Ex Taq Kit (Takara). The primers specific for the target mRNAs/miRNAs were obtained from Wcgene Biotech (Shanghai, China). Since hypoxia has an influence on GAPDH expression, β-actin and U6 were used to evaluate mRNA and miRNA levels, respectively, in cell samples.(16). For EV samples, expression levels were normalized to those of the external control cel-miR-39 (RiboBio, Guangzhou, China). The results are shown as relative expression levels calculated by the 2−ΔΔCT method.
3.6. Vector construction, lentivirus production and cell transfections
The LV2-hsa-miR-340-5p-mimic vector and LV2-hsa-miR-340-5p-inhibitor vector were constructed as lentiviral vectors (GenePharma, Shanghai, China). Cells were infected at 40-50% confluence and selected with puromycin to confirm stable transfection. pcDNA3.1 vectors containing KLF10 and the negative control vectors were purchased from GenePharma. siRNAs and mimics were purchased from RiboBio. Lipofectamine 3000 reagent (Invitrogen) was used for transfection. The sequences of the vectors are listed in Table S1.
3.7. 5-Ethynyl-2ʹ-deoxyuridine (EdU) assay
We evaluated cell proliferation using an EdU assay kit (RiboBio). Twenty-four hours after coculture with EVs, cells were digested and seeded into 24-well plates (2 × 104 cells/well) and cultured with RPMI-1640 medium (10% FBS) for 24 hours before EdU (50 μmol/L) was added. According to the protocol, cells were then incubated for 2 hours at 37°C, fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature. After washing with PBS, 1× ApolloR reaction cocktail was added to react with the EdU for 30 minutes. Subsequently, Hoechst 33342 was added for 30 minutes to stain nuclei. Images of cells were acquired under a Nikon microscope. Proliferation was assessed using the mean number of cells in three fields per sample.
3.8. Luciferase reporter assay
Sequences corresponding to the 3ʹ-UTR of KLF10 mRNA and containing the wild-type or mutated miR-340-5p binding sequence were synthesized by GeneScript (Nanjing, China). We inserted these sequences into the XbaI and SacI sites in the pmirGLO dual-luciferase miRNA target expression vector (Promega, USA). These vectors were cotransfected with the miR-340-5p-mimic/miR-340-5p-inhibitor or miR-NC by using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Luciferase activity was measured with a Luciferase Reporter Assay System (Promega).
3.9. RNA immunoprecipitation (RIP)
An EZ-Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore,) was used to conduct the RIP assay. Cells were collected and lysed in pre-cooled lysis buffer supplemented with 1 mM PMSF, protease inhibitor and RNase inhibitor. Cells were then incubated with RIP buffer containing magnetic beads conjugated to a human anti-Argonaute 2 (Ago2) antibody (Millipore) or normal IgG (Millipore). The precipitate was digested, and the co-immunoprecipitated RNA was then isolated for PCR.
3.10. Colony formation assay
We seeded 200, 400, 800, 2000 and 5000 cells/well in 6-well plates one day before irradiation with 0, 2, 4, 6 or 8 Gy. After two weeks, each well was washed with PBS for three times, fixed with 4% paraformaldehyde for 30 minutes and finally stained with crystal violet. Colonies were counted macroscopically. The survival curve was constructed using a single-hit, multi-target model.
3.11. Apoptosis analysis
Apoptosis was assessed using an apoptosis detection kit (Vazyme, Nanjing, China) following the manufacturer's instructions. At 72 hours post irradiation with 8 Gy, cells were digested, resuspended in 500 μl of binding buffer with 5 μl of annexin V-FITC solution and 5 μl of propidium iodide (PI), and stained at room temperature for 15 minutes in the dark. Flow cytometry (FACScan; BD Biosciences, USA) and FlowJo software (BD, USA) were used to analyze the cells.
3.12. Immunofluorescence assay
A total of 5×104 cells were seeded into a confocal laser dish one day before irradiation with 8 Gy. Four hours post irradiation, cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes and permeabilized with 0.1% Triton X-100 for 2 hours. Cells were then blocked with 5% BSA for 90 minutes and washed with PBS. After incubation with the anti-γ-H2AX primary antibody (1:400; Cell Signaling Technology) overnight at 4℃, cells were washed with PBS and incubated with an Alexa Fluor 555-conjugated secondary antibody (Beyotime) for 90 minutes. Cells were washed with PBS, treated with DAPI staining solution for 20 minutes and observed using a confocal fluorescence microscope (Leica).
3.13. Nude mouse xenograft model
This study was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-1901012). BALB/c nude mice were purchased from the Animal Center of Nanjing Medical University. A total of 2×106 cells were injected subcutaneously into the flanks of the nude mice. Tumours were measured with calipers every 5 days, and the tumour volume was calculated using the following formula: volume = (width2 × length)/2. When the tumour volume reached 50 mm3, 10 µg (50 µl) of EVs or 50 µl of PBS was injected into the tumour once daily for 5 days. In addition, tumours were irradiated in an RS 2000 Pro X-Ray Bio-irradiator (Radsource, USA) with a dosage of 2 Gy per day for 4 consecutive days starting on the second day of EV injection. Two weeks post IR, the mice were euthanized. In addition, we injected NC-Te13 cells and miR-340-5p mimic Te13 cells into the flanks of nude mice to study the effect of miR-340-5p. When the tumour volumes reached 50 mm3, four fractions of radiation (2 Gy/f) were applied to the tumours, and the mice were sacrificed 14 days after the radiation exposure was completed. Lead shields were used to prevent radiation injury.
3.14. Immunohistochemistry
Immunohistochemistry was performed as previously reported(17). The staining intensity was scored on a four-grade scale: 0 (no staining), 1 (weak staining), 2 (intermediate staining), or 3 (strong staining). The staining percentages were divided into 4 grades: 0 (no positive cells), 1 (<25% positive cells), 2 (25%-50% positive cells) and 3 (>50% positive cells). The product of the staining intensity and staining percentage was used as the final staining score.
3.15. TUNEL assay
The TUNEL assay was performed using an In Situ Cell Death Detection Kit (Roche, Germany). Following the manufacturer’s protocols, sections were deparaffinized and rehydrated. Antigen retrieval was performed using hot 0.1 M citrate buffer (pH 6.0), and incubated with the TUNEL reaction mixture (containing TdT and fluorescein-conjugated dUTP) for 1 hour at 37℃. DAPI was used to stain nuclei. Apoptotic cells were analysed using a fluorescence microscope (Leica). For the negative control reaction, TdT was not included in the reaction mixture.
3.16. Statistical analysis
All experiments in this study were carried out in triplicate. Differences between groups were determined using the non-parametric Kruskal-Wallis test or Mann-Whitney U test with the Bonferroni’s correction. A chi-squared test was used to detect differences in clinical data. The patients were divided into high expression and low expression groups based on the median gene expression level. Kaplan-Meier analysis was used to compare recurrence in OSCC patients. STATA 14.0, SPSS 22.0 and GraphPad Prism 8.0 software were used to perform statistical analysis, and a p value <0.05 was considered statistically significant (*p<0.05, **p<0.01, ***p<0.001).