A total of 84 patients diagnosed with NSCLC at the PLA Rocket Force Characteristic Medical Center between January 2014 and April 2016 were enrolled in the present study. The diagnosis of all patients was confirmed by cytology or histopathology assessment. The inclusion criteria were as follows: Complete medical records, physical examination, fibrobronchoscopy, chest, head and abdomen CT or abdominal ultrasound scan, whole-body bone scan, final pathological diagnosis and clear surgical histopathological diagnosis. The exclusion criteria were as follows: Neurological abnormality, severe dysfunction of the heart, liver, kidney or other organs, diabetes and hypertension. The patient clinicopathological characteristics are presented in Table I. Parallel information was collected from 42 age- and sex-matched patients who did not exhibit abnormalities upon physical examination at the PLA Rocket Force Characteristic Medical Center in May 2014. The routine laboratory and imaging tests performed included complete blood count, baseline electrolyte measurement, blood chemistry profiling and chest X-rays.

All patients were followed up by outpatient visits or telephone between June 2016 and June 2018, and no patients were lost to the follow-up. The date of diagnosis was set as the start of follow-up, and survival time was assessed monthly. The period between the date of diagnosis and death due to any cause was considered as the overall survival time. Patients who passed away due to non-neoplastic causes and those who were alive at the end of follow-up were included as censored data that met statistical requirements.

The human bronchioloalveolar lung carcinoma cell line A549 was obtained from the cell bank of Peking Union Medical College Hospital. A549 cells were cultured at 37°C in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; Gibco; Thermo Fisher Scientific, Inc.) in a humidified chamber at 5% CO₂. Cells were seeded at 5×10⁵ cells per 60-mm culture dish.

Following 24-h culture, A549 cells in the exponential growth phase were irradiated with 2 or 4 Gy at a rate of 442.89 cGy/min in a linear accelerator (source distance, 100 cm; 6 MeV; Elekta Instrument AB). The A549 cells treated with each dose of radiation were divided into 3 subgroups and cultured for 2, 12 or 24 h. Non-irradiated A549 cells were collected at the indicated time points and used as negative controls.

A total of 30 female athymic BALB/c nu/nu mice (6–8 weeks; weight, ~18 g) were obtained from Vital River Laboratories and housed in specific pathogen-free conditions with free access to food and water, temperature of 20–25°C, relative humidity of 55–65% and a 12-h light/dark cycle in the Beijing Laboratory Animal Research Center (Beijing Institute of Science and Technology, Beijing, China). Mice were injected with 5×10⁵ A549 cells irradiated with 0-, 2- or 4-Gy X-rays via the tail vein, and were separated into the 0-, 2- and 4-Gy groups. The mice in the 0-Gy group were divided into 3 groups according to the time point of observation, with 6 mice in each group, and mice that did not undergo inoculation of A549 were used as controls. Mice were sacrificed 3, 6 or 10 weeks after inoculation, and lung and sera samples were harvested and stored at −80°C.

Total RNA were extracted from the serum of patients and nude mice using a mirVana™PARIS™ Kit (cat. no. 1556; Ambion; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Briefly, ≤625 µl serum was mixed with an equal volume of 2X denaturing solution at room temperature and incubated on ice for 5 min. Subsequently, an equal volume of acid-phenol: Chloroform was added for extraction of the aqueous phase.

The cells were washed twice with PBS prior to the addition of 600 µl lysis/binding buffer (mirVana miRNA Isolation kit; cat. no. 1561; Ambion; Thermo Fisher Scientific, Inc.) directly to the culture plate or flask. The lysates were manually harvested using a sterile cell scraper and transferred to a 2-ml tube. The samples were stored at −80°C unless RNA was immediately extracted according to the manufacturer's instructions (Ambion; Thermo Fisher Scientific, Inc.).

For murine lung tissue, 0.2 g tissue was ground into a powder in liquid nitrogen with a pre-cooled mortar and pestle. The tissue was mixed with 1 ml lysis/binding buffer, followed by acid-phenol: Chloroform extraction. Ethanol was added to the samples prior to passing them through a filter cartridge containing a glass-fiber filter for RNA immobilization. The filter was washed with miRNA wash solution 1/2/3 three times, and the RNA was eluted with 30 µl elution solution (low ionic-strength solution), with both solutions included in the aforementioned kit.

To quantify miRNA expression in A549 cells, the TaqMan miRNA assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) was performed according to the manufacturer's instructions. The template cDNA was synthesized from total miRNA using a TaqMan™ MicroRNA Reverse Transcription kit (cat. no. 4366596; Applied Biosystems; Thermo Fisher Scientific, Inc.) and quantified by qPCR using the following parameters: Hold at 50°C for 2 min, denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 60 sec. The TaqMan^® Universal PCR Master mix II (cat. no. 4440038; Applied Biosystems; Thermo Fisher Scientific, Inc.) was used with a LightCycler 480 real-time PCR system (Roche Molecular Systems, Inc.). The primers and probes of the three miRNAs were purchased from Thermo Fisher Scientific, Inc. (TaqMan^® microRNA assay; cat. no. 4427975; Applied Biosystems; Thermo Fisher Scientific, Inc.) U6 RNA was used for internal normalization. Relative expression was calculated using the following equations: Relative gene expression=2^−∆∆Cq; -∆∆C_q=(C_{q gene of interest}-C_{q internal control gene})_treated -(C_{q gene of interest}-C_{q internal control gene})_untreated (6,28).

A total of 5×10⁵ A549 cells were seeded per 60-mm plate 18–24 h prior to the transfection. The medium was replaced with antibiotic-free medium 6–12 h before transfection. Synthetic miRNA mimics and inhibitors (miR-130a, miR-25 and miR-191*) and the negative control (cat. nos. 4464084, 4464066, 4464058 and 4464076; Thermo Fisher Scientific, Inc.) were transfected into 70–80% confluent A549 cells using the Lipofectamine^® RNAiMAX (cat. no. 13778030; Invitrogen; Thermo Fisher Scientific, Inc.) reagent according to the manufacturer's instructions. The prepared mixtures of Lipofectamine^® RNAiMAX and miRNA mimics/inhibitor diluted in Opti-MEM (cat. no. 31985-062; Invitrogen; Thermo Fisher Scientific, Inc.) were incubated separately for 5 min at room temperature, then added to adherent A549 cells and incubated at 37°C for 24 h. After 24 h transfection, cells were harvested for the extraction of total RNA and protein.

Cell invasion was measured using 24-well micro chemotaxis chambers with a membrane pore size of 8.0 µm (EMD Millipore) pre-coated with 10 µg/ml Matrigel at 37°C for 30 min (BD Biosciences). A549 cells transfected with miRNA mimics or inhibitors were resuspended in 100 µl serum-free DMEM (cat. no. 10566016; Gibco; Thermo Fisher Scientific, Inc.) and seeded into the upper well of each chamber. A total of 100 µl DMEM containing 10% FBS and 10% fibronectin (BD Biosciences) was loaded into the lower well.

Cells were incubated at 37°C for 22 h. Cells in the upper well that had not migrated were scraped off using a cotton swab, and the cells that had migrated through the membrane were fixed with 4% paraformaldehyde for 20 min and stained with Giemsa for 10 min, both at room temperature. Fixed cells were imaged under a phase-contrast microscope (magnification ×400) for six fields and counted for statistical analysis.

Transfected A549 cells were washed twice in ice-cold PBS and lysed in RIPA lysis buffer with protease inhibitors (50 nmol/l HEPES, pH 7.5, 150 nmol/l NaCl, 1% glycerol, 1% Triton, protease inhibitor cocktail). The protein concentration was determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). A total of 25 µg protein was loaded per sample for SDS-PAGE (12% acrylamide) and transferred to polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked using 5% skimmed milk in TBS-Tween (TBST; 0.05% Tween-20) at room temperature for 1 h, and then incubated with antibodies against VEGF (1:800; cat. no. 34-4300; Sigma-Aldrich; Merck KGaA), CCR-7 (1:2,000; cat. no. ab32527; Abcam) or tubulin (1:2,000; cat. no. SC-12462; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature, the membrane was washed 3 times with TBST (10 min each), followed by incubation with horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody for 1 h at room temperature (1:1,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.). The signal was captured using a Super Signal West Pico chemiluminescent substrate (cat. no. 34080; Pierce; Thermo Fisher Scientific, Inc.) to visualize the bands, and Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) was used for grayscale analysis.

Data are presented as the mean ± SD. Data were processed using SPSS version 19.0 (IBM Corp.). The Mann-Whitney U test was used to determine the statistical difference between the expression levels of the three miRNAs and different clinical characteristics. Associations between clinical characteristics and miRNA expression were assessed by logistic regression analysis, and survival was analyzed using the Kaplan-Meier method with Cox regression. One-way ANOVA followed by Dunnett's post hoc test was used to evaluated the statistical differences of the expression levels of the three miRNAs or VEGF or CCR-7 mRNA/protein expression at different time points of A549 cells with 0-, 2- and 4-Gy X-ray irradiation or with miRNA transfection. Kruskal-Wallis test followed by Tukey's or Dunnett's post hoc tests was used to evaluate the statistical differences of three miRNAs expression in lung tissues and serum of nude mice injected with A549 cells with different doses of X-ray irradiation. Receiver operating characteristic curves were used to evaluate the diagnostic value of miR-130a, miR-25 and miR-191* in patients with NSCLC. P<0.05 was considered to indicate a statistically significant difference.

A total of 84 patients (58 male and 26 female) with lung cancer with a mean age of 60.78 years (range, 37–87) were enrolled in the present study. According to the International Association for the Study of Lung Cancer Staging Project (29), various clinical stages were present among the enrolled patients, including three cases at stage I, five cases at stage III and 76 cases at stage IV. Detailed clinical characteristics of the patients are presented in Table I. Notably, the expression of miR-25 and miR-191* was significantly higher in patients treated with radiotherapy compared with those who did not receive radiotherapy treatment, in patients with SCC compared with those with ADC, and in patients who succumbed to NSCLC or the associated complications during the follow-up period compared with those who survived (P<0.05 and P<0.01; Table I; Fig. S1B and C). miR-130a expression levels were significantly different between the radiotherapy and non-radiotherapy groups (P=0.004; Fig. S1A), and there was also a significant difference in miR-25 expression between patients aged ≥60 and <60 years (P=0.035; Table I; Fig. S1B). There was no association between miR-130a, miR-25 or miR-191* expression and sex, smoking status, lymph node metastasis, extracranial metastasis, brain metastasis (BM), number of BMs, maximum diameter of BM, Tumor-Node-Metastasis (TNM) T stage (30), node (N) stage (30) or Eastern Cooperative Oncology Group (ECOG) (31,32) performance status (P>0.05; Table I).

Based on the optimal cut-off values for miR-130a, miR-25 and miR-191* (Data S1 and Fig. S2), logistic regression analysis was performed to determine the association between the expression of each miRNA and clinical characteristics. The results indicated that miR-130a expression was associated with radiotherapy (P=0.021), but not sex, age, smoking status, histological type, survival status, lymph-node metastasis, BM, extracranial metastasis or ECOG value. By contrast, miR-25 and miR-191* expression levels were associated with radiotherapy (P=0.026 and P=0.014, respectively), histological type (P=0.010 and P=0.005, respectively) and survival status (P=0.020 and P=0.043, respectively) (Table II). The expression of miR-25 was also significantly associated with age (P=0.019).

Kaplan-Meier survival curve analysis demonstrated that patients exhibiting high expression of miR-25 and miR-191* presented with low survival rates compared with patients with low expression of the two miRNAs (P=0.036 and P=0.009, respectively; Fig. 1B and C). The median survival time of patients in the high miR-25 and miR-191* expression groups was 40 and 36 months, respectively, which was notably lower compared with the survival times of patients in the low expression groups [63 (P=0.036) and 65 (P=0.009) months, respectively; Table III]. Multivariate Cox survival analysis indicated that radiotherapy (P=0.024), ECOG status (P=0.008) and miR-191* expression (P=0.034) were independent prognostic factors for NSCLC in this cohort (Table IV).

The aforementioned results indicated that radiotherapy, histological type and prognosis were associated with the expression of miR-130a, miR-25 and miR-191* in patients with NSCLC. Therefore, the expression levels of miR-130a, miR-25 and miR-191* in A549 cells treated with different doses of irradiation were examined in vitro and in vivo. At 2 h post-2-Gy irradiation, the expression levels of miR-130a and miR-25 were significantly increased in A549 cells (P<0.05 and P<0.01), whereas the expression levels of miR-191* were significantly increased at 12 h post-irradiation compared with those in the respective control groups (Fig. 2A). At 2 h post-4-Gy irradiation, the expression levels of miR-191* and miR-25 were also significantly increased (P<0.05 and P<0.01), whereas the expression levels of miR-130a were significantly increased at 12 h post-irradiation compared with those in the respective control groups (P<0.05) (Fig. 2A).

For the in vivo study, 6 mice each in the 0-Gy group were sacrificed at 3, 6 and 10 weeks, and 5 mice each in the 2- and 4-Gy groups were sacrificed at 10 weeks after injection. At 3, 6 and 10 weeks after A549 cell injection, miR-130a and miR-25 expression was significantly increased in mice serum samples, but there was no significant increase in lung tissue expression of these miRNAs compared with in the control mice. The expression levels of miR-191* were increased in the serum of nude mice at 3 and 10 weeks after injection when the mice exhibited visible tumors scattered in the lungs (data not shown), while the miR-191* expression in the lung tissues of nude mice was upregulated at 10 weeks after injection compared with that of the control group (P<0.05; Fig. 2B). Both 2- or 4-Gy irradiation resulted in significant upregulation of the expression levels of all three miRNAs at 10 weeks post injection in the serum and lung tissue compared with those in the control group (P<0.01; Fig. 2C). Notably, the expression levels of miR-130a and miR-25 in the lung tissues of mice treated with 2- and 4-Gy irradiation exhibited a dose-dependent tendency to increase compared with those of mice injected with non-irradiated A549 cells (P<0.05 and P<0.01; Fig. 2C).

To delineate the roles of the three miRNAs in response to X-ray irradiation, the effects of miR-130a, miR-25 and miR-191* on the invasive capability of A549 cells were assessed in vitro following transfection with miRNA mimics and inhibitors. Transfection efficiency was verified by qPCR (data not shown). The results of the cell invasion assay demonstrated that the invasiveness of A549 cells transfected with miR-130a, miR-25 and miR-191* mimics was significantly increased compared with that of the control group (P<0.05 and P<0.01; Fig. 3A). Additionally, transfection of A549 cells with miR-130a and miR-191* inhibitors significantly reduced invasiveness (P<0.05; Fig. 3B), while transfection of A549 cells with miR-25 inhibitor had no significant effect on cell invasiveness.

Our previous study has demonstrated that 2- and 4-Gy X-rays promote the invasion of A549 cells in vitro and in vivo mainly by upregulating the expression of VEGF and CCR-7 mRNA (33). Following A549 cell exposure to 2- and 4-Gy X-ray irradiation, the expression of miR-130a was positively correlated with the expression of VEGF and CCR-7 mRNA (P<0.05), while the expression of miR-25 and miR-191* was not significantly correlated with the expression of VEGF/CCR-7 mRNA (P>0.05) (34). Therefore, the effects of miR-130a, miR-25 and miR-191* on the expression of VEGF and CCR-7 in X-ray-irradiated A549 cells were subsequently investigated. Upregulation of VEGF expression levels was observed following transfection of all three miRNA mimics compared with those in the control groups (P<0.05 and P<0.01; Fig. 4A), whereas upregulation of CCR-7 expression was observed in the miR-25 mimics group at 72 h and in the miR-191* mimics group at 24, 48 and 72 h post-transfection (P<0.05 and P<0.01; Fig. 4A). Compared with those in the negative control group, protein expression levels of VEGF were upregulated at 48 and 72 h post-transfection with miR-130a mimics (P<0.05 and P<0.01, respectively; Fig. 4B), whereas the expression of CCR-7 was upregulated only at 72 h post-transfection with miR-130a mimics (P<0.05; Fig. 4B). By contrast, no significant changes in the mRNA (Fig. 4A) or protein expression levels (data not shown) of VEGF or CCR-7 were observed in the three miRNA inhibitor transfection groups.