Patients and samples
A total of 318 NSCLC patients were recruited between March 2019 to July 2019 from Shandong Cancer Hospital, Affiliated to Shandong First Medical University (Jinan, China). None of NSCLC patients have received treatment, including surgery, radiotherapy and chemotherapy. Clinical and pathological characteristics of patients, including age, gender, smoking, TNM stage, (according to the eighth edition American Joint Committee on Cancer) were obtained from the patients’ records. In addition, a total of 312 healthy donors, who did not have any other tumor disease, were acquired from the same hospital. Exosomes from 5 healthy donors and 10 NSCLC patients were subjected to miRNA array, and then 318 patients and 312 controls were subjected to verification. The peripheral blood from patients and healthy donors were collected and then RT-PCRcentrifuged at 1, 000× g for 10 min at 4°C. The serums were then transferred to fresh tubes and stored at -80°C for exosome isolation.
Isolation of Exosomes
Serum exosomes were isolated using ultracentrifugation according to the procedures as described previously [20, 23]. Briefly, the serum was thawed on ice and centrifuged at 10, 000×g for 30 min at 4°C to remove the cellular debris. Then 1 mL of supernatant was ultracentrifugated (Beckman Coulter, Brea, CA, USA) at 100, 000× g for 2 h at 4°C to pelletize exosomes. Exosome pellets were characterized with qNano, TEM, and western immunoblotting, followed by miRNA sequencing and RNAs extraction.
qNano assay
The size and distribution of exosomes isolated from serum were analyzed using the Tunable Resistive Pulse Sensing (TRPS) on the qNano (Izon Science Ltd, Christchurch, New Zealand) according to the manufacturer’s instructions. Data were analyzed by software (Izon Control Suite version 3.3.2.2001; Izon Science).
Transmission Electron Microscopy (TEM) assay
15 µL of exosome samples were added on formvar-coated copper grid. After 1 min, the remaining liquid was removed by filter paper and the samples were placed in 15µL of 2% uranyl acetate staining solution for 1 min at room temperature. Subsequently, excess liquid was wiped off, and the copper grid was baked under the lamp for 10 min, followed by observing the exosomes by FEI Tecnai T20 transmission electron microscope (FEI Company, USA).
Western immunoblotting
Exosomes or cellular proteins were separated using 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocked with 5% evaporated skimmed milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h, the membranes were probed with rabbit primary antibodies against CD63, TSG101, and GM130 overnight at 4oC. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The immunoreactive bands were visualized by ECL blotting detection reagents (Bio-Rad, USA).
MiRNA profiling and Data Analysis
Qualified RNA from exosomes was used to label miRNA by the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) according to the manufacturer’s instruction. The Hy3™-labeled samples were hybridized on to the miRCURYTM LNA Array (v.19.0) (Exiqon) according to the instruction manual. Afterwards, the wash buffer kit (Exiqon) was used to wash the slides for several times, followed by scanning with the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA). Images were uploaded into GenePix Pro 6.0 software (Axon) for extracting data and aligning the grid. MiRNAs, with intensities >=30, were selected to calculate the normalization factor. After normalizing the expressed data, miRNAs, those were significant differentially expressed by fold change >=2.0, were filtered. MiRNA expression levels between the samples were distinguished by hierarchical clustering.
RNA isolation and Real-Time PCR (RT-PCR)
All processes were carried out in an RNase-free area. Total RNAs were isolated from serum exosomes using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer’s instructions. Then, the Mix-X miRNA First Strand Synthesis Kit (TaKaRa Bio, Nojihigashi, Kusatsu, Japan) was used to reverse-transcribe RNA into complementary DNA (cDNA) according to the manufacturer’s protocol. RT-PCR was performed using TB-Green Premix Ex Taq II Reagent (TaKaRa Bio) according to the manufacturer’s instructions on a Roche LightCycler480 System (Roche Diagnostics, Basel, Switzerland) [22, 24]. The relative expression levels of miRNAs were calculated using the following equation: ΔCT = CtmiRNA-CtU6 as described previously [25]. Each sample was analyzed in duplicate.
Statistical Analysis
SPSS 22.0 (IBM, Ehningen, Germany) and GraphPad Prism 6.0 (San Diego, CA, USA) were used for statistical analysis. The different expressions of miRNAs among groups were determined using the Mann-Whitney unpaired test or paired t test. The association between miRNAs and the clinical characteristics was evaluated by χ2 test or paired-samples t test. Receiver operating characteristic (ROC) and area under the curve (AUC) ware applied to assess the diagnostic power of the candidate predictors. p < 0.05 was considered to be statistically significant.