Specimens
Fresh and formalin-fixed paraffin-embedded (FFPE) tumour tissues and matched adjacent normal epithelium were obtained from 58 surgically treated patients; 23 of them were diagnosed with ITACs, 15 with SIPs, and 20 had documented NIPs. All samples were collected from the archive of the Pathological Anatomy Unit of the Hospital University of Ancona, and from the Otorhinolaryngology Department of Budrio-Metropolitan Hospital Bologna, Italy. The clinical data included information on gender, age, histology, staging according to the TNM classification (UICC 2009), therapeutic protocol and the follow-up conditions.
Whole blood was collected from ITAC patients (n = 13) at the time of clinical examination and serum was prepared. The control group consisted of 19 healthy subjects recruited at the clinical of Occupational Medicine, Polytechnic University of Marche, Ancona, Italy. None of them had ever been occupationally exposed to wood and leather dust, as documented by their occupational histories.
RNA, circulating MiR-126 isolation and quantitative RT-PCR
Total RNA from cultured cells and tissue samples (10–100 μg) was obtained using the RNeasy Mini Kit (Qiagen) and the RecoverAll total nucleic acid isolation kit (Ambion), respectively, according to the manufacturer’s instructions. MiR-126, IRS1, VEGF-A, SOX-2 and EGFL7 first-strand cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative RT-PCR (qPCR) was performed using the TaqMan Gene Expression Master Mix (Applied Biosystems) with U6 and GAPDH as the housekeeping gene for the MiR and the genes, respectively. qPCR assays were performed using the Mastercycler EP Realplex instruments (Eppendorf) and results expressed as relative level (2-ΔCT), and fold changes (2-ΔΔCT). For circulating MiR-126 detection, RNA was isolated from 250 μl of diluted serum (1:10), or from the exosomal fraction (20 μg protein) and MiR-126 was detected as previously described [17, 18].
Bisulphite modification and genomic sequencing
The methylation status of CpG dinucleotides close to the EGFL7 T2 promoter was analysed. The bisulphite sequencing assay was performed using 1 mg of bisulphite-treated genomic DNA from malignant tissue and adjacent non-cancerous tissue of seven patients with SNC. Bisulphite conversion was performed using the EZ DNA Methylation™ Kit (Zyno Research, Euroclone) according to the manufacturer’s instructions. Fragments of interest were amplified using the following specific primer pairs designed with the Primer3 software, i.e. forward: 5′-TGA TTT AGT GAT TTC GGT GAG G-3′; reverse, 5′-AAC CCT TTA CTA ACT TTC AAA CCC-3′. PCR products were gel-purified by means of the Wizard SV gel and PCR Clean-up kit (Promega) or the FastGene Gel/PCR Extraction Kit (Nippon Genetics), and sequenced using the Reverse primer (5′-AAC CCT TTA CTA ACT TTC AAA CCC-3′) to analyse the DNA methylation status. Sequencing of purified PCR products was carried out using automated DNA sequencers at Eurofins MWG Operon (Germany). All sequences were visualized with BioEdit Sequence Alignment Editor 7 [19] and aligned with the ClustalW option included in this software.
Cell culture
Malignant nasal squamous cell carcinoma from the pleural effusion (MNSC, RPMI 2650) and fibroblasts (IMR-90) were obtained from the ATCC and grown in the RPMI-1640 and DMEM medium, respectively, with 10% FBS, 1% penicillin and 10% streptomycin (Life Technologies). Human umbilical vein endothelial cells (HUVECs) obtained from GIBCO (Life Technologies) were grown in Medium 200 with the large vessel endothelial supplement (LVES, Life Technologies). Cells were maintained at 37 °C and 5% CO2, and cultured for not more than six passages within 1 month after resuscitation and periodically checked for the absence of mycoplasma contamination using the PCR Mycoplasma Test. Cell authentication was performed using the PowerPlex Fusion 6C System (Promega, Fitchburg, WI).
Exosome isolation and exosome uptake
Exosomes were isolated from HUVECs cultured in exosome-depleted serum-containing medium as previously described [18]. For exosome uptake assay, isolated exosomes were stained with PKH-67 (20 μM, 4 min; Sigma), a probe used to label lipids on membrane surface of exosomes. After washing in PBS, PKH-26-labelled exosomes were placed in conditioned medium of MNSC cells, and uptake was periodically analysed by flow cytometry (FACS Calibur, BD). Alternatively, uptake was assessed after 4 h incubation by fluorescent microscopy (Axiocam MRc5 Zeiss).
Ectopic MiR-126 expression
MNSC cells (2 × 104 per well in a 24 well plate) were stably transfected with the pCMV-MiR plasmid carrying the MiR-126 sequence 5’-UCG UAC CGU GAG UAA UAA UGC G-3′ (OriGene) using the TransIT-LT1 reagent (Mirus). Selection of transfected cells was performed using G418 (0.6 mg/ml; Sigma) added to the cell culture medium after transfection. G418-resistant clones were analysed for MiR-126 expression and maintained in the RPMI media with 0.6 mg/ml G418. Alternatively, cells were transiently transfected with the MiR-126 mimetic (MISSION® microRNA Mimic; Sigma) using High Perfect Transfection reagent (Qiagen). MiR-126 function was blocked with the anti-sense oligonucleotide 5’-GCA UUA UUA CUC ACG GUA CGA-3′ (IDT); scrambled sequences were used as a control.
Acid vesicles and red oil O staining of lipids
Acid vesicles (AVs) in MNSC cells and their MiR-126-transfected counterparts were evaluated by acridine orange (AO) staining. Cells were seeded on coverslips in a 6-well plate, allowed to attach overnight, incubated with 5 mg/ml AO for 30 min at 37 °C. For detection of lipid droplets (LDs), cells grown on coverslips were fixed in 70% (v/v) cold ethanol and stained with the Oil Red O solution in 60% (v/v) isopropanol. Cells were analysed using fluorescent and optical microscopy, respectively (Zeiss, Axiocam MRC5; magnification 40× or 60×).
Migration and cell proliferation assay
Migration assays were performed after seeding cells in the 12-well plate allowed to reach confluence and treated with mitomycin-C (5 μg/ml, 3 h; Sigma) to block the cell cycle. Cells were wounded by scratching with sterile pipette tip and supplemented with exosomes isolated from HUVECs (20 μg/ml). Migration was quantified as wound-healing percentage of the cells determined by the ratio of the ‘wound width’ at 72 h time and the wound width at 0 h. The MTT assay (Sigma) was performed and Ki67-positive cells were estimated as a measure of cell proliferation. MNSC cells and the MiR-126 plasmid- or mimetic-transfected cells (3 × 104 cells/well in 96-well-plate) were incubated at different time points and cell viability evaluated. Alternatively, cell viability was analysed in MNSC cells exposed to exosomes isolated from HUVECs. Ten microliters of MTT (5 mg/ml) was added to each well and after 3 h incubation, the crystals prodused were dissolved in isopropanol and absorbance read at 550 nm in an ELISA plate reader (Sunrise, Tecan). The results were expressed as relative change with respect to the controls set as 100%. Proliferation was assessed in permeabilized cells using the anti-Ki67 IgG and fluorescence microscopy (Zeiss; Axiocam MRc5, magnification 60×). The proliferation index was expressed as a percentage of Ki67-positive cells.
Glucose uptake and glucose, ATP and lactate detection
MNSC cells were seeded in 96-well black-bottom plates (3 × 104 cells per well) in low glucose (1 g/l) DMEM at 5% CO2 and 37 °C. After overnight incubation, the cells were treated with 2-nitrobenzodeoxyglucose (2-NBDG, 50 μM) for 30 min. The level of fluorescence intensity was evaluated at 550/590 nm using a fluorescence plate reader (Infinite F200 PRO, Tecan). Glucose, lactate and intracellular ATP were evaluated in the presence and absence of rotenone (20 μM, 5 h) and 2-deox-glucose (2DG; 5 mM, 5 h) using commercial kits (Abcam) according to the manufacturer’s protocol. The results were normalized to the total protein content.
Mitochondrial reducing activity
Mitochondrial reducing activity (MRA) was assessed in MiR-126-transfected MNSC cells and their parental counterparts as the reduction of resazurin based on the mitochondrial metabolic activity [15, 20]. Cells were incubated with resazurin (6 μM) and fluorescence intensity read at 0–240 min in a fluorescence plate reader (Infinite F200 PRO). The results were normalized to the total protein detected using the Bradford assay (Sigma).
Colony-forming assay
MNSC cells and their MiR-126 plasmid-transfected counterparts were seeded in 0.35% low melting point agar overlaid with 0.7% low melting point agar in 24-well plates and cultured at 37 °C and 5% CO2 for 1 month. Every 7 days, 0.5 ml of fresh medium was added to each well, the number of colonies counted, and the size and the shape evaluated after 3 weeks. In addition, MNSC colonies in soft-agar were treated with exosomes from HUVECs (20 μg/ml) each week, and their size and shape evaluated after a 3-week treatment. Alternatively, MNSC cells (103 cells per well in 24-well plate) were treated with the MiR-126 mimetic (100 nM), and colony formation was evaluated. Colonies were fixed with formalin (4.0% v/v), stained with crystal violet (0.5% w/v), and counted using a stereomicroscope.
Triple co-culture model
A 3-μm trans-well insert (Costar 3452; Corning,) was first plated with 105 cultured IMR90 cells in an inverted position. After 6 h of incubation, inserts were flipped over and placed into a six-well trans-well plate, where 3 × 105 HUVEC cells were loaded on the other side of the insert and cultured for 24 h. This HUVEC-IMR90 pre-coated trans-well inserts were then placed into another six-well trans-well plate, where 2 × 105 malignant nasal-septum carcinoma (MNSC) cells had been plated. Exosome-exposed HUVECs (50 μg/ml) were added to the triple co-culture system, and after 48 h of incubation MNSC cells were collected.
Western blot analysis
Cells were lysed in the RIPA buffer containing Na3VO4 (1 mM) and protease inhibitors (1 μg/ml). The cell lysate proteins were separated using SDS-PAGE and transferred onto nitrocellulose membranes (Protran). After blocking with 5% non-fat milk in PBS-Tween (0.1%), the membranes were incubated with antibodies against IRS1 (Bethyl), phospho-p38 MAPK, p38-MAPK, phospho-ERK1/2 and ERK1/2 (all Cell Signaling). β-actin was used as a loading control. After incubation with the HRP-conjugated secondary IgG (Sigma), blots were developed using the ECL detection system (Pierce Biotechnology). The band intensities were visualized and quantified with ChemiDoc using the Quantity One software (BioRad Laboratories).
Statistical analysis
Data are expressed as mean values ± standard deviation (SD). Comparisons among groups of data were performed using one-way analysis of variance (ANOVA), with Tukey post-hoc analysis. A p-value ≤0.05 was considered significant. All statistical analyses were performed using the SPSS software.