A total of 15 cases of paraffin-embedded NSCLC tissue (including 8 cases of squamous carcinoma and 7 cases adenocarcinoma; mean age, 58.7 years; age range, 33–75 years; 10 cases ≥60 years old and 5 cases <60 years old; 9 males and 6 females) and 8 cases non-cancerous normal tissue confirmed by a lung needle biopsy were collected from patients without previous radiotherapy or chemotherapy between January 2016 and December 2016 who underwent treatment in the Department of Oncology of the First People's Hospital of Lianyungang (Lianyungang, China). The exclusion criteria included: Patients who had received chest radiotherapy or systemic chemotherapy prior to sampling; previous history of oncology; participation in other drug clinical trials and anti-tumor treatments; pregnant and lactating women; and known history drug abuse (except alcohol abuse). All the samples were from human lung biopsy tissue and confirmed by experienced pathologists who were blinded to the study. The present study was approved by the ethics committee of the First People's Hospital of Lianyungang. Written informed consent was obtained from the participating individuals.

The human NSCLC cancer cell line Calu-1 (human squamous cell carcinoma) (17), H358 (human lung adenocarcinoma, #SCSP-583) (18), H460 (human lung large-cell carcinoma) (19), H292 (human lung mucoepidermoid carcinoma-lymph node metastatic strain) (20), H1650 (human lung adenocarcinoma) (21), A549 (human lung adenocarcinoma) (22), H1975 (human lung adenocarcinoma) (21), and H1299 (human lung adenocarcinoma lymph node metastatic strain) (23) were obtained from the Cell Bank of Shanghai Institute of Biological Science, Chinese Academy of Science (Shanghai, China). Among these cell lines, Calu-1 was cultured with McCoy's 5A medium containing 10% (v/v) fetal bovine serum and 1% antibiotics, while other cell lines were maintained as monolayers when cultured in cell culture flasks with RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% antibiotics. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂. All the cell culture medium and additives were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All 8 cell lines were used for expression analysis miR-497 by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), whereas Calu-1 and H1975 cells were further employed for the cell proliferation, cell migration, cell apoptosis, cell radiosensitivity, miR-497 and KDR interaction analysis, and the in vivo tumor formation experiment.

miR-497 mimic and miR-NC were obtained from the Sangon Biotech Co., Ltd. (Shanghai, China) and the detailed information regarding the sequence were from previous description (24). Cells were grown to a confluence of ~40% and transfected with 50 nM miR-mimic and miR-NC encapsulated with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in Gibco Opti-MEM™ I Reduced Serum medium (cat. no. 31985-062; Gibco; Thermo Fisher Scientific, Inc.) for 48 h. All the experiments were repeated for at least 3 times.

Total NSCLC tissue RNA or cellular RNA of human NSCLC cancer cell lines were extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT-qPCR for kinase insert domain receptor (KDR) was conducted using an One Step SYBR^® PrimeScript™ RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) and an iQ5 Real-time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR conditions were 1 cycle for 30 sec at 95°C, 40 cycles of 5 sec at 95°C and 34 sec at 60°C, followed by 1 cycle for 15 sec at 95°C and 60 sec at 60°C. Expression of the GAPDH gene was assessed simultaneously in all samples as an internal control. For miRNA RT-qPCR, Taqman^® MicroRNA Reverse Transcription and Taqman MicroRNA Assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) kits were employed for experiments. The expression of U6 was used as the internal control. Relative gene expression was determined with the 2^−ΔΔCq method (25). Oligonucleotide primers specific for KDR and GAPDH were obtained from Sangon Biotech Co., Ltd. The sequences of the PCR primers used are as follows: KDR, 5′-CCGTCAAGGGAAAGACTACG-3′ (forward) and 5′-AGATGCTCCAAGGTCAGGAA-3′ (reverse); miR-497, 5′-GTGCAGGGTCCGAGGT-3′ (forward), and 5′-TAGCCTGCAGCACACTGTGGT-3′ (reverse); and U6, 5′-GCTTCGGCACATATACTAAAA-3′ (forward), and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse).

Human NSCLC cancer cell lines Calu-1 and H1975 (3×10³ cells) were seeded in 96-well plates in complete medium [Calu-1 cells were cultured with McCoy's 5A medium containing 10% (v/v) fetal bovine serum and 1% antibiotics, H1975 cells were maintained as monolayers when cultured in cell culture flasks with RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% antibiotics] and transfected with miR-497 mimic and miR-NC, as aforementioned. After 2 days at 37°C, 5% CO_2., cell proliferation during a time period of consecutive 4 days was evaluated by Cell Counting Kit-8 method (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer's protocols, using a microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA) to measure the absorbance.

Human NSCLC cancer cell lines Calu-1 and H1975 (800 cells) were seeded in 6-well plates in complete medium [Calu-1 cells were cultured with McCoy's 5A medium containing 10% (v/v) fetal bovine serum and 1% antibiotics, H1975 cells were maintained as monolayers when cultured in cell culture flasks with RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% antibiotics] and transfected with miR-497 mimic and miR-NC, as aforementioned. After medium replacement [Calu-1 cells were cultured with McCoy's 5A medium containing 10% (v/v) fetal bovine serum and 1% antibiotics, H1975 cells were maintained as monolayers when cultured in cell culture flasks with RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% antibiotics] at 48 h post-transfection, the cells were irradiated with 2 Gy X ray to efficiently induce apoptosis within 48 h, and then harvested and suspended for the assay. Subsequently, ~1×10⁷ cells were washed in PBS, resuspended in Annexin V and propidium iodide (PI) staining solution containing 5 µl Annexin V, 10 µl PI and 195 µl binding buffer (Annexin V-FITC/PI Double Dyeing Cell Apoptosis Test kit; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) and incubated at room temperature for 20 min under the dark condition, and then analyzed immediately with a flow cytometer (BD FACS Aria) by BD FACSDiva software version 4.0 (BD Biosciences; Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Human NSCLC cancer cell lines Calu-1 and H1975 (800 cells) were seeded in 6-well plates in complete medium [Calu-1 cells were cultured with McCoy's 5A medium containing 10% (v/v) fetal bovine serum and 1% antibiotics, while other cell lines were maintained as monolayers when cultured in cell culture flasks with RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% antibiotics] and transfected with miR-497 mimic and miR-NC, as aforementioned. After medium replacement [Calu-1 cells were cultured with McCoy's 5A medium containing 10% (v/v) fetal bovine serum and 1% antibiotics, H1975 cells were maintained as monolayers when cultured in cell culture flasks with RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% antibiotics] at 48 h post-transfection, the cells were irradiated with a series dose of 0–8 Gy X-rays and maintained at 37°C in a humidified atmosphere containing 5% CO₂ for 12 days, when they were stained with crystal violet at room temperature for 1 h. The colony survival (a colony was defined as ≥50 cells) was counted under a light microscope (×40; ECLIPSETs2; Nikon Corporation, Tokyo, Japan). The whole process was performed three times to obtain the mean number of colony formations.

A Transwell system was employed to perform the cell invasion assay. Briefly, resuspended Calu-1 and H1975 cells [Calu-1 cells were cultured with McCoy's 5A medium containing 10% (v/v) fetal bovine serum and 1% antibiotics, H1975 cells were maintained as monolayers when cultured in cell culture flasks with RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% antibiotics] (2×10⁵ cells) transfected with miR-497 mimic and miR-NC, as aforementioned, were seeded into the upper chamber prefilled with Matrigel, and the lower chamber was added with RPMI-1640 medium supplemented with 20% FBS. After the transwell plate was maintained in a routine cell culture incubator for a specific period of time of 48 h, the upper chamber was retained and the membranes were obtained for hematoxylin staining at room temperature for 1 min. The cell number of each membrane was determined in 3 randomly picked fields (×200 magnification) under a light microscope. All the experiments were performed in triplicate.

Cells obtained from the aforementioned treatment (routine cell culture or after transfection with miR-497 mimic and miR-NC) were lysed in radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology, Beijing, China), followed by high-speed centrifugation (15,000 × g for 5 min at 4°C) and protein quantification using a bicinchoninic acid assay. Cellular proteins (20 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Following blocking in 5% non-fat milk containing PBS at room temperature for 2 h, the membranes were incubated with anti-VEGF receptor 2 (VEGFR2) monoclonal primary antibodies (1:1,000 dilution; Cell Signaling Technology, Inc. Danvers, MA, USA; #9698) at room temperature for 1 h. GAPDH (1:1,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; #sc-32233) was used at room temperature for 1 h as the loading control. Appropriate horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG, HRP-conjugated antibody; 1:2,000 dilution; incubated at room temperature for 1 h; Cell Signaling Technology, Inc.; cat. no. 7074) were applied to detect labeled proteins. The protein bands were developed with a SuperSignal Ultra Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.) on X-ray films (Kodak, Rochester, NY, USA).

The possible miR-497 binding sites in KDR gene 3′-UTR were predicted using bioinformatics software (Targetscan version 7.1; http://www.targetscan.org/vert_71/). The predicted and mutated sequences targeting on KDR 3′-UTR were amplified and cloned into a pMIR-REPORT Dual-Luciferase miRNA Target Expression Vector (Promega Corporation, Madison, WI, USA; named pMIR-REPORT-KDR-WT and pMIR-REPORT-KDR-MUT, respectively). Subsequently, Calu-1 and H1975 cells were co-transfected with 0.5 µg pMIR-REPORT-KDR-WT or pMIR-REPORT-KDR-MUT vectors, and 100 nM negative control (NC) miRNA or miRNA-mimics (Shanghai GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 2000. The luciferase activities were measured using a Dual-Luciferase Reporter Assay kit (Promega Corporation) 48 h after transfection. A control reporter was co-transfected to provide an internal control for normalizing the activity of the experimental reporter.

Recombinant lentiviral particles expressing miR-497 (5′-CAGCAGCACACUGUGGUUUGUA-3′) or scramble control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) were obtained from Shanghai GenePharma Co., Ltd. Cells were grown to 40% confluence and transduced with complete medium [Calu-1 cells were cultured with McCoy's 5A medium containing 10% (v/v) fetal bovine serum and 1% antibiotics, and H1975 cells were maintained as monolayers when cultured in cell culture flasks with RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% antibiotics] containing lentiviral particles expressing miR-497 or scramble control siRNA at concentrations of 1×10⁸ transducing units/ml [multiplicity of infection (MOI) of 20] (18) at 37°C for 48 h. Polybrene (cat. no. H9268; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a concentration of 8 µg/ml was added simultaneously to increase infection efficiency. No adverse effects were observed on the cell viability by siRNA or Polybrene (data not shown). The siRNAs had no off-target effects, and did not affect cell adherence, shape and viability at the MOI of 20, according to manufacturer's protocol and treatment duration. The cells were used at 48 h after infection.

Athymic nude (nu/nu) mice (male:female, 1:1 ratio; total 8 mice) at 6 weeks old (20–22 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China), housed in an air-conditioned room at 22°C with a 12/12 h light/dark cycle and 40–60 % humidity and free access to food and water. The tumors were generated by subcutaneous injection of 2×10⁶ miR-497 or scramble control siRNA lentivirus particles infected Calu-1 cell suspended in 50 µl PBS into the dorsal region near the thigh. Mice were then weighed and assessed for tumor size every three day by measuring tumor length and tumor width. Furthermore, the body weight of the mice was also determined every 5 days. At week 4 post-treatment, all the mice were sacrificed by cervical dislocation and the tumors were excised, weighed and imaged. The study protocol was approved by the Institutional Animal Care and Use Committee of the First People's Hospital of Lianyungang, which is adherent to the generally accepted international guidelines for animal experimentation (26).

All statistical analyses were conducted using SPSS v18 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA). Data are presented as the mean ± standard deviation. The unpaired Student's t-test or one-way analysis of variance followed by Tukey's post hoc test was used to examine differences between groups. Dose-response cell survival curves were fitted to a multitarget model, S=1-(1-e-D/D0)^N. S, ratio of survival tumor cells at a given dose; D, a given dose; D0, the dose increment that reduces the cell survival to 37% of the initial value depicted on the exponential portion of the curve; N, back extrapolation of the exponential portion of the survival curve to zero dose. P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the role of miR-497 in NSCLC, the expression level of miR-497 was examined in 15 NSCLC tissues and 8 non-cancerous adjacent normal tissues, and the results demonstrated that significantly decreased levels of miR-497 expression in NSCLC, compared with normal tissues (0.47±0.27 vs. 1.03±0.27; P<0.05; Fig. 1A). Furthermore, the expression of miR-497 and KDR in 8 NSCLC cancer cell lines was also examined, and the results demonstrated that KDR and miR-497 were significantly decreased in H1299 and H1975 cells, compared with in other cell lines, respectively (Fig. 1B; P<0.05). Additionally, H1975 and Calu-1 cell lines with reduced expression of miR-497 were further employed for the following experiments.

In order to observe the effects of miR-497 on the cell behavior of Calu-1 and H1975 cells, miR-497 overexpression was performed on Calu-1 and H1975 cells. As depicted in Fig. 2A, a 6.7 and 5.9 times expression level increase was exhibited in miR-497-mimic transfected Calu-1 and H1975 cells, compared with mimic-NC-transfected cells, respectively. For cell proliferation, a significantly decreased cell proliferation was observed on days 3 and 4 in miR-497-mimic-transfected Calu-1 and H1975 cells, compared with mimic-NC transfected cells (Fig. 2B and C). Furthermore, a significantly decreased cell invasion was also observed in miR-497-mimic-transfected Calu-1 (50±3.5 vs. 157±3.7; P<0.001) and H1975 (48±2.4 vs. 153±4.2; P<0.001) cells, compared with mimic-NC-transfected cells (Fig. 2D). These results indicated that miR-497 overexpression decreases cell proliferation and migration in Calu-1 and H1975 cells.

The effects of miR-497 overexpression on the cell apoptosis and radiosensitivity in Calu-1 and H1975 cells was further evaluated. The results demonstrated that there was significantly increased cell apoptosis following 2 Gy radiation in miR-497-mimic-transfected Calu-1 and H1975 cells, compared with mimic-NC-transfected cells (Fig. 3A; P<0.001). Furthermore, the clone formation was consistently decreased after 2, 4, 6 and 8 Gy radiation, and the sensitization enhancement ratio in Calu-1 and H1975 were 1.48 and 1.58, respectively (Fig. 3B and C), and miR-497 significantly increased the radiosensitivity of these cells. These results indicated the enhanced radiosensitivity following miR-497 transfection.

In order to investigate the mechanism of miR-497 in NSCLC cell lines Calu-1 and H1975, TargetScan was searched and it was determined that KDR may be the potential target gene of miR-497 (Fig. 4A). Subsequently the KDR wild-type and mutated plasmids were constructed, and a luciferase assay was performed to confirm its interaction with miR-497. The results demonstrated that miR-497-mimic transfection significantly decreases luciferase activity in KDR wild-type, compared with mimic-NC transfected cells, while no difference was determined on the cells transfected with KDR mutated plasmid following miR-497-mimic or mimic-NC transfection (Fig. 4B). Furthermore, it was determined that there is significantly decreased expression of KDR in miR-497-mimic-transfected Calu-1 and H1975 cells, compared with mimic-NC-transfected cells (Fig. 4C). Additionally, it was also observed that there is a significantly decreased level of VEGFR2 in miR-497-mimic-transfected Calu-1 and H1975 cells, compared with mimic-NC-transfected cells (Fig. 4D).

Due the tumor-promoting effects of miR-497 observed in vitro, ectopic tumor nude mice models were established to test the effects of miR-497 in vivo. As depicted in Fig. 5, miR-497 knockdown human lung squamous carcinoma cell line Calu-1 exhibited a decreased level of tumor growth in vivo according to the tumor size and weight. These results indicated that miR-497 acts as a negative regulator on tumor growth in vivo.