A549 cells and A549/cisplatin (A549/DDP) cells (Guangzhou Zixiutang Biotechnology Co., Ltd., Guangzhou, China) were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. All cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

For miR-146a target prediction, PicTar (http://pictar.mdc-berlin.de/), miRBase (http://www.mirbase.org/), miRanda (http://www.microrna.org/microrna/home.do), Bibiserv (https://bibiserv.cebitec.uni-bielefeld.de/agenda) and TargetScan (http://www.targetscan.org/vert_71/) were used to identify and analyze potential targets of JNK2.

Total RNA was extracted from culture cells with guanidine thiocyanate, 2-mercaptoethanol and chloroformin GenElute Mammalian Total RNA Purification kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 3 min on ice and the RNA concentration was read using the NanoDrop 2000 spectrophotometer. RNA quality was validated using a Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). Total RNA (100 ng) was used for cDNA synthesis. Subsequently, cDNA was labeled with fluorescence, fragmented and hybridized to the Affymetrix GeneChip Gene 1.0 ST Human Array (Thermo Fisher Scientific, Inc.). Following incubation at 45°C for 16 h, the arrays were washed with 1X saline sodium citrate (SSC), 0.1% SDS, 0.1X SSC, 0.1% SDS and 0.1X SSC and water for 2 min of each at room temperature, and then scanned (Affymetrix Model 3000 scanner; Thermo Fisher Scientific, Inc.). The data adjustments included data filtering, log₂ transformation, gene centering. In addition, the signal was normalized using a Locally-weighted Regression filter.

Total RNA was isolated with buffer RLT and purified from A549 and A549/DDP cells using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) for 5 min on ice. miRNA was prepared using the miRcute miRNA isolation kit (Tiangen Biotech Co., Ltd., Beijing, China) and cDNA was synthesized from the RNA by reverse transcription employing the Qiagen OneStep RT-PCR kit (Qiagen GmbH, Hilden, Germany). Sequences of all the primers are presented in Table I. qPCR amplification was performed to enable the fluorescence-based quantitation of gene expression using the SYBR Green Quantitative RT-PCR kit (Sigma-Aldrich; Merck KGaA). U6 was regarded as the reference gene. The PCR volume and conditions were set up as previously described (28) The primers for miR-182-5p (catalog no., MIRAP00211), miR-106b (catalog no., MIRAP00129) and miR-146a (catalog no., MIRAP00183) were from the MystiCq^® microRNA qPCR Assay Primer purchased for Sigma-Aldrich (Merck KGaA). In additon, 2^−∆∆Cq (29) was applied for gene quantification. The primer sequences of U6 were: Forward, 5′-AAAGCAAATCATCGGACGACC and reverse, 3′-GTACAACACATTGTTTCCTCGGA.

miR-146a mimic and inhibitor targeting JNK2 (1 ng) were synthesized by GenePharma, Inc. (Sunnyvale, CA, USA). While, the A549/DDP cells infected with empty plasmid were used as controls (MOCK group). The sequences are presented in Table I. The transfection in A549/DDP cells was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol (23).

A549/cisplatin (A549/DDP) cells (1×10⁴ cells/well) were seeded in 96-well plates in RPMI-1640 medium. An MTT assay was performed using a Cell proliferation kit I (GE Healthcare Life Sciences, Little Chalfont, UK) according to the manufacturer's protocol. Subsequently, absorbance was determined at 570 nm using VersaMax (Molecular Devices; Thermo Fisher Scientific, Inc.) to estimate MTT-formazan production dissolved with dimethyl sulfoxide after 24, 48 and 72 h incubation. The OD490 was detected using a spectrodensitometer under 490 nm wavelength.

The FITC Annexin V Apoptosis Detection kit with PI (BioLegend, Inc., San Diego, CA, USA) was used for cell apoptosis assay. After 72 h incubation, the transfected A549/cisplatin (A549/DDP) cells (1×10⁵) were fixed with 2.5% glutaraldehyde for 30 min at room temperature. Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining flow cytometry was used to determine apoptotic rates, by identifying the relative amount of Annexin V-FITC positive and PI negative cells. Unstained cells and cells stained with Annexin V-FITC or PI alone were used as controls. PI staining flow cytometry was used to determine the cell cycle. CYTOSPEC™ version 7.0 from Purdue University Cytometry Laboratories was applied for data analysis.

The cell invasion assay was performed using 24-well Transwell plates, as previously described (30). A total of 8×10⁴ cells were seeded in serum-free RPMI-1640 medium in the upper and lower Transwell chambers and incubated for 24 h. Matrigel (BD Biosciences) was pre-coated on the upper side of the membrane and incubated at 37°C for 1 h for gel formation. Following the removal of the Transwell inserts, the cells that had not migrated through the filter and remained inside each Transwell were removed by wiping with a cotton swab. Cells that had migrated through the filter and adhered to the other side of the filter were stained with crystal violet at room temperature for 5 min for image capture, and counted using a light microscope with ×200 magnification (30).

Human JNK2 cDNA, containing putative and mutant target sites for miR-146a, was chemically synthesized and inserted into a pMIR-REPORT™ vector (Ambion; Thermo Fisher Scientific, Inc.). For the luciferase reporter assay, miR-146a mimic, miRNA mimic control, miR-146a inhibitor and miRNA inhibitor control were transfected into wild-(pMIR-jnk2-wt) or mutant-type (pMIR-jnk2-mut) reporter vectors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Luciferase activity was determined 48 h after transfection using a dual-luciferase assay kit (Promega Corporation, Madison, WI, USA) (31). Additionally, Renilla luciferase activity was applied for luciferase intensity normalization.

The total protein was extracted from A549/DDP cells and separated using SDS-PAGE (10% gel), as previously described (32). Subsequently, the gel was transferred to a polyvinylidene difluoride membrane (Solvay Chemicals, Brussels, Belgium) and blocked with 5% skim milk at room temperature for 1 h. The rabbit anti-JNK2 (catalog no., PA528664), -p53 (catalog no., PA527822) and -B cell lymphoma (Bcl-)2 primary antibodies (catalog no., PA520069) purchased from Wuhan Khayal Bio-Technology Co., Ltd. (Wuhan, China) were used at a dilution of 1:1,000. The incubation with primary antibodies was at room temperature for 1 h. Subsequently, the goat anti-rabbit immunoglobulin G secondary antibody conjugated with horseradish peroxidase (cat. no. Ab97051, Abcam, Cambridge, MA, USA) was used at a dilution of 1:10,000. In addition, the incubation with secondary antibodies was at room temperature for 45 min. The signal was visualized using the enhanced chemiluminescence kit (GE Healthcare Life Sciences). Image J 1.41 software (NIH, Bethesda, MD, USA) was used to compare the gray values between the proteins of interest and the internal control protein (β-actin), and between the phosphorylated protein and the total protein.

The expression levels of JNK2, p53 and Bcl-2 mRNA in A549/DDP cells were assessed using RT-qPCR. Total RNA was extracted from the cells using the TRIzol method (Thermo Fisher Scientific, Inc.) at 4°C for 15 min. Subsequently, cDNA was synthesized from the RNA by reverse transcription using SYBR Green Quantitative RT-PCR kit (Sigma-Aldrich; Merck KGaA). PCR amplification was performed to enable fluorescence-based quantitation of gene expression. PCR reaction volumes were 10 µl and composed of cDNA (1 µl), primers (0.2 µl each), 2X Premix Ex Taq (5 µl) and H₂O (3.6 µl). The primer sequences used are presented in Table I. For cDNA synthesis, samples were incubated at 40°C for 30 min, 98°C for 5 min and 5°C for 5 min. The PCR conditions were as follows: Pre-denaturation at 96°C for 5 min, followed by initiation at 94°C for 30 sec, annealing at 60°C for 30 sec and elongation at 78°C for 1.5 min for 35 cycles, after which samples were stored at 4°C. Additionally, 2^−∆∆Cq (29) was applied for gene quantification. β-actin was selected as the internal reference.

All data are presented as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA) via Student's t-test. For all comparisons, P<0.05 was considered to indicate a statistically significant difference.

miRNA microarray analyzed 30 miRNAs and PicTar, miRBase, miRanda, Bibiserv and TargetScan were used to identify and analyze potential targets of JNK2. A total of 3 miRNAs, including miR-146a, miR-182-5p and miR-106b, were identified to be potential targets of JNK2 in >2 databases. In addition, the expression levels of these 3 miRNAs in A549 and A549/DDP cells were determined. As presented in Fig. 1, the expression levels of miR-182-5p, miR-106b and miR-146a were demonstrated to be downregulated in NSCLC A549/DDP cells, compared with A549 cells. Only the expression of miR-146a revealed statistical significance (P<0.05).

The luciferase reporter assay was performed to determine whether miR-146a directly targets JNK2 by binding to the 3′-UTR of the JNK2 gene. Wild-type and mutant JNK2 3′UTR sequences, which contained the miR-146a binding site, were constructed and inserted into vectors (Fig. 2A). The reporter construct and miR-control, miR-146a mimics, miR-146a inhibitor and relative controls were transfected into the A549/DDP cell line. As presented in Fig. 2B, the luciferase reporter activity represented a significant decrease in JNK2 3′-UTR in the presence of miR-146a mimics in A549/DDP cells, compared with the miR-control. Furthermore, a significant increase was observed in the presence of miR-146a inhibitor (Fig. 2B). Therefore, JNK2 was identified as a direct target of miR-146a.

To investigate the effects of miR-146a on the cisplatin sensitivity, the A549/DDP cells were transfected with miR-146a mimics and inhibitors. The A549/DDP cells infected with empty plasmid were used as controls (MOCK group). The proliferation of cells was determined after 24, 48 and 72 h incubation, and the results are presented in Fig. 3. After 24 h incubation, upregulated miR-146a A549/DDP cells revealed a significantly decreased proliferation rate, compared with the other two groups. The cell apoptosis and cell cycle were determined using flow cytometry and are presented in Figs. 4 and 5. Fig. 4A, B and C present cell apoptosis in the mock, mimic and inhibitor groups, respectively, and Fig. 4D demonstrates the apoptosis rate of A549/DDP cells. The results of the present study revealed that, in the miR-146a overexpression group, the apoptosis rate was significantly increased compared with the other two groups. The relative proportion of A549/DDP cells in G₁, S and G₂ period were calculated and are presented in Fig. 5. Cells in the miR-146a mimic group were arrested in the S period, which resulted in cells failing to enter the G₂ period and complete mitosis. For invasion, the results of the Transwell assay of infected A549/DDP cells are presented in Fig. 6. The relative invasion rate of A549/DDP cells in the miR-146a mimic group was significantly decreased, compared with the other two groups.

Expression levels of JNK2, P53 and Bcl-2 mRNA and proteins in infected A549/DDP cells were determined using RT-qPCR and western blot analysis (Fig. 7). JNK2, as the direct target of miR-146a, was identified to be significantly upregulated by miR-146a overexpression. Additionally, p53 was demonstrated to be significantly upregulated by miR-146a overexpression, whereas Bcl-2 was revealed to be significantly downregulated by miR-146a.