NSCLC cancer tissues and healthy adjacent tissues (≥5 cm from the tumor margin) were obtained from 50 patients (age, 32–70 years; mean age, 45±7 years; 32 male patients and 18 female patients) who were diagnosed with NSCLC in Jingmen No. 1 People's Hospital between February 2018 and April 2019. In all patients with NSCLC, diagnosis was histopathologically confirmed. Written informed consent was obtained from all patients. The present study was approved by Jingmen No. 1 People's Hospital Ethics Committee (approval no. JM2018010232).

Human bronchial epithelium 16HBE cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. NSCLC lines (A549, SK-MES-1, H1975 and SK-LU-1) were purchased from American Type Culture Collection. Cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in humidified air with 5% CO₂ at 37°C.

To explore the effects of NORAD on lung cancer cells, cells were divided into the following groups: i) Control (cells without treatment); ii) pc-control (cells transfected with pc-control); iii) pc-NORAD (cells transfected with pc-NORAD); iv) small interfering RNA (si)-control (cells transfected with si-control); and v) si-NORAD (cells transfected with si-NORAD). To explore the potential mechanisms underlying NORAD in lung cancer cells, cells were divided into the following groups: i) Control (cells without treatment); ii) mimic-control (cells transfected with miR-422a mimic-control); iii) mimic (cells transfected with miR-422a mimic); iv) pc-NORAD; v) mimic + pc-NORAD; vi) inhibitor-control (cells transfected with miR-422a inhibitor-control); vii) inhibitor (cells transfected with miR-422a inhibitor); viii) si-NORAD; and ix) inhibitor + si-NORAD.

SK-MES-1 cells were transfected with pc-NORAD, pc-control, miR-422a mimic (5′-ACUGGACUUAGGGUCAGAAGGC-3′; Shanghai GenePharma Co., Ltd.) or mimic control (5′-UUUGUACUACACAAAAGUACUG-3′; Shanghai GenePharma Co., Ltd.). A549 cells were transfected with si-NORAD (5′-AAGCCACCTTTGTGAACAGTA-3′), si-control (5′-TTCTCCGAACGTGTCACGT-3′), miR-422a inhibitor (5′-GCCUUCUGACCCUAAGUCCAGU-3′) or inhibitor control (5′-CAGUACUUUUGUGUAGUACAAA-3′). Cell transfection (2×10⁵ cells/well) was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h post-transfection, subsequent experiments were performed.

Total RNA was extracted from cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.). The following temperature protocol was used for reverse transcription: 37°C for 40 min and 85°C for 5 min. Subsequently, NORAD expression levels were determined by qPCR using a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and SYBR Green PCR Master Mix (Toyobo Life Science). The sequences of the primers used to measure NORAD expression levels were as follows: NORAD forward, 5′-CCTGGAAGGTGAGCGAAGT-3′ and reverse, 5′-AGAGGGTGGTGGGCATTT-3′; and GAPDH forward, 5′-TGGTCACCAGGGCTGCTT-3′ and reverse, 5′-AGCTTCCCGTTCTCAGCC-3′. To detect miR-422a expression levels, qPCR was performed using the SYBR Premix Ex Taq kit (Takara Bio, Inc.). The sequences of the primers used to measure miR-422a expression levels were as follows: miR-422a forward, 5′-ACUGGACUUAGGGUCAGAAGGC-3′ and reverse, 5′-GCCUUCUGACCCUAAGUCCAGU-3′; and U6 forward, 5′-CTTCGGCAGCACATATAC-3′ and reverse, 5′-GAACGCTTCACGAATTTGC-3′. The following thermocycling conditions were used for qPCR: Pre-degeneration at 95°C for 30 sec; 39 cycles at 95°C for 10 sec and 60°C for 30 sec; and final extension at 72°C for 30 sec. mRNA and miRNA expression levels were quantified using the 2^−ΔΔCq method (17) and normalized to the internal reference genes GAPDH and U6, respectively.

Cells (1×10³/well) were plated in 96-well plates for 24, 48 or 72 h at 37°C. Subsequently, 10 µl CCK-8 reagent was added to each well and incubated for 1 h 37°C. Cell viability was measured by detecting the absorbance at a wavelength of 450 nm using a microtiter plate.

Cell migration was assessed by performing a wound healing assay. During the wound healing assay, cells (2×10⁵ cells/well) were cultured in RPMI-1640 medium supplemented with 1% FBS at 37°C with 5% CO₂. At 90% confluence, a single scratch was made in the cell monolayer using a medium-sized pipette tip. The monolayer was washed with PBS to remove cell debris. At 0 and 24 h, images of the cells were captured using a light microscope to measure the scratch width (magnification, ×100). Migration rate=(scratch width at 0 h-scratch width at 48 h)/scratch width at 0 h.

At 48 h post-transfection, cells (3×10⁵) were cultured in medium without serum. A Transwell assay (pore size, 8 µm; Corning, Inc.) was performed to detect cell invasion. Cells (2×10⁵ cells/ml) were plated into the upper chamber with medium containing 1% FBS, which was pre-coated with Matrigel^® at 37°C for 30 min. Medium supplemented with 10% FBS (500 µl) was plated in the lower chambers. Following incubation for 24 h at 37°C, cells on the upper chamber surface were removed. Invading cells were fixed with 50% methanol for 30 min at 4°C and stained with 0.1% crystal violet for 30 min at room temperature. Stained cells were visualized under a light microscope (magnification, ×100) and the number of invading cells was calculated to determine the relative invasion rate.

starBase (starbase.sysu.edu.cn) was used to predict the target gene of NORAD, and the binding sites were verified by performing adual-luciferase reporter assay. pGL3-NORAD 3′-UTR wild-type (wt) plasmid and pGL3-NORAD 3′-UTR mutated (mut) plasmid were obtained from Guangzhou RiboBio Co., Ltd. In brief, the wt 3′-UTR of NORAD containing the miR-422a binding sequences was obtained by PCR amplification from human genomic DNA, and mutated (mut) NORAD obtained using the Quick-Change Site-Directed Mutagenesis kit (Stratagene). The wt and mut 3′-UTRs of NORAD were inserted into the pGL3 luciferase reporter plasmids (Promega Corporation) to obtain pGL3-NORAD 3′-UTR wt plasmid and pGL3-NORAD 3′-UTR mut plasmid, respectively. 293T cells (2×10⁵ cells; The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences) were co-transfected with NORAD-wt plasmid (100 ng) or NORAD-mut plasmid (100 ng), and 50 nM miR-422a mimic or 50 nM miR-422a mimic-control using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. At 48 h post-transfection, luciferase activity was measured using the dual-luciferase reporter assay system (Promega Corporation). Firefly luciferase activities were normalized to Renilla luciferase activities.

Total protein was extracted from cells using RIPA lysis buffer (Santa Cruz Biotechnology, Inc.) and cell lysates were centrifuged at 20,000 × g for 10 min at 4°C. Equal amounts of protein (30 µg) were separated via 10% SDS-PAGE and transferred onto PVDF membranes at 100 V for 1.5 h. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies targeted against: Matrix metallopeptidase (MMP)2 (1:1,000; 72 kD; cat. no. ab37150; Abcam), MMP9 (1:1,000; 95 kD; cat. no. ab73734; Abcam), E-cadherin (1:10,000; 97 kD; cat. no. ab40772; Abcam), N-cadherin (1:1,000; 130 kD; cat. no. ab18203; Abcam) and GAPDH (1:10,000; 36 kD; cat. no. ab181602; Abcam). Following primary incubation, the membranes were incubated with a secondary horseradish peroxidase-conjugated antibody (goat anti-rabbit IgG; 1:10,000; cat. no. ab205718; Abcam) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence detection reagent (Thermo Fisher Scientific, Inc.). Protein expression was quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.) with GAPDH as the loading control.

Statistical analyses were performed using SPSS software (version 20.0; IBM Corp). Data are presented as the mean ± standard deviation. Comparisons between two groups were analyzed using the paired or unpaired Student's t-test. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. The correlation between continuous variables was analyzed using Pearson's correlation analysis. P<0.05 was considered to indicate a statistically significant difference. All experiments were performed at least three times.

The expression levels of NORAD in NSCLC tissues and cells were detected via RT-qPCR. The results indicated NORAD expression was significantly higher in NSCLC tissues compared with healthy adjacent tissues (Fig. 1A). Moreover, NORAD expression levels in NSCLC cell lines (A549, SK-MES-1, H1975 and SK-LU-1) were significantly higher compared with the normal human bronchial epithelial 16HBE cell line (Fig. 1B).

The RT-qPCR results indicated that pc-NORAD and si-NORAD were successfully transfected into SK-MES-1 and A549 cells, respectively (Fig. 1C and D). CCK-8, wound healing and Transwell assays were performed to detect the effects of NORAD on NSCLC cell viability, migration and invasion, respectively. At 48 and 72 h, pc-NORAD-transfected cells displayed higher viability compared with the pc-control group, whereas si-NORAD-transfected cells displayed significantly lower viability compared with the si-control group (Fig. 1E and F). Moreover, the results indicated that NORAD overexpression significantly reduced the number of cells in G₁ phase and increased the number of cells in G₂/M phase compared with the pc-control group. By contrast, NORAD knockdown increased the number of cells in G₁ phase and reduced the number of cells in G₂/M phase compared with the si-control group (Fig. 1G and H). In addition, the wound healing assay demonstrated that pc-NORAD significantly increased cell migration compared with the pc-control group, whereas si-NORAD displayed the opposite results compared with the si-control group (Fig. 2A and B). Similarly, the results of the Transwell assay indicated that NORAD-overexpression cells displayed a significantly higher invasion rate compared with the pc-control group, whereas NORAD-knockdown cells displayed a significantly decreased invasion rate compared with the si-control group (Fig. 2C and D).

The potential mechanisms underlying NORAD in NSCLC cells were explored, and the relationship between NORAD and miRNA was predicted. starBase was used to identify the binding sites between NORAD and miR-422a (Fig. 3A). A dual-luciferase reporter assay indicated that the luciferase activity of NORAD-wt was significantly reduced by miR-422a mimic compared with the blank group, which verified the relationship between NORAD and miR-422a (Fig. 3B and C). Furthermore, the expression of miR-422a in NSCLC tissues was detected via RT-qPCR. The results demonstrated that miR-422a expression levels were significantly lower in NSCLC tissues compared with healthy adjacent tissues (Fig. 3D). In addition, miR-422a expression levels in the NSCLC cell lines (A549, SK-MES-1, H1975 and SK-LU-1) were significantly reduced compared with 16HBE cells (Fig. 3E). Pearson's correlation analysis indicated that miR-422a expression was negatively correlated with NORAD expression in NSCLC tissues (Fig. 3F).

CCK-8, wound healing and Transwell assays were performed to detect the effects of NORAD and miR-422a on cell viability, migration and invasion. The CCK-8 assay results indicated that miR-422a overexpression significantly reduced cell viability compared with the mimic-control, but cells transfected with miR-422a mimic and pc-NORAD displayed significantly increased cell viability compared with the mimic group. However, miR-422a knockdown significantly increased cell viability compared with the inhibitor-control, which was reversed by co-transfection with si-NORAD (Fig. 4A and B). Moreover, the wound healing assay results suggested that miR-422a mimic significantly decreased cell migration compared with the mimic-control group, whereas miR-422a inhibitor displayed the opposite effect compared with the inhibitor-control group. Furthermore, cells co-transfected with miR-422a mimic and pc-NORAD displayed significantly increased cell migration compared with the mimic group, whereas cells co-transfected with miR-422a inhibitor and si-NORAD displayed decreased cell migration compared with the inhibitor group (Fig. 4C and D). The results of the Transwell assay revealed that miR-422a overexpression significantly inhibited cell invasion compared with the mimic-control group, but miR-422a knockdown displayed the opposite effects compared with the inhibitor-control group. However, alterations to NORAD expression levels partly reversed the effects of miR-422a on cell invasion (Fig. 4E and F).

The RT-qPCR results indicated that the expression levels of miR-422a in SK-MES-1 cells were significantly decreased by NORAD overexpression compared with the mimic + pc-NORAD group, whereas NORAD knockdown displayed the opposite effects compared with the inhibitor + si-NORAD group (Fig. 5A and B). To explore the effect of NORAD on NSCLC cell EMT, western blotting was performed to measure the expression levels of EMT-related proteins, such as MMP2, MMP9, E-cadherin and N-cadherin. The results demonstrated that miR-422a overexpression significantly reduced the protein expression levels of MMP2, MMP9 and N-cadherin, but significantly increased the protein expression levels of E-cadherin compared with the mimic-control group. NORAD overexpression reversed miR-422a overexpression-mediated effects on protein expression (Fig. 5C). However, miR-422a knockdown significantly increased the protein expression levels of MMP2, MMP9 and N-cadherin, and significantly reduced the expression levels of E-cadherin compared with the inhibitor-control group. NORAD knockdown reversed miR-422a knockdown-mediated effects on protein expression (Fig. 5D).