A549 and A549/DDP human lung cancer cell lines were used in the present study. The cells were cultured in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies) and penicillin (100 U/ml; Invitrogen Life Technologies), at 37°C in an atmosphere containing 5% CO₂. Blood samples (n=27) were collected from patients with advanced lung cancer that had received cisplatin-based chemotherapy between May 2010 and December 2012, at the Affiliated Hospital of Jiangnan University (Wuxi, China). The median patient age was 64 (range, 49–85 years). The patients included 16 females and 11 males. All specimens were histologically classified by a professional pathologist, according to the national NCCN guidelines for NSCLC version 3.2011 (16) in a blinded manner. Written informed consent was obtained from all of the patients recruited. The methodologies of the present study conformed to the standards set by the Declaration of Helsinki; and the study was approved by the Ethical Committee of the Affiliated Hospital of Jiangnan University (Wuxi, China)

The sequence of Meg3 was synthesized by Genewiz (Suzhou, China). All recombinant lentiviruses were produced by calcium phosphate-mediated transient transfection of HEK293T cells, according to standard protocols. Briefly, HEK293T cells from Clontech were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% FBS and penicillin/streptomycin (100 U/ml). The subconfluent cells in a 10 cm culture dish were co-transfected with lentiviral vector (10 µg), the lentiviral packaging vectors pLv-GFP or pLv-Meg3 (2 µg), pMDLg/pRRE (5 µg), and the vesicular stomatitis virus G glycoprotein (VSVG) expression vector pMD2G (3 µg). The viruses were collected from the culture supernatants on days 2 and 3 post-transfection. The cultured cells were incubated with lentivirus at various concentrations for 24 h and cultured with DMEM, containing 10% FBS. A pure population of infected cells were sorted based on the GFP-expression by flow cytometry and >98% of the cells were GFP-positive following sorting. The plasmid was subcloned into a lentiviral vector, and then co-transfected into HEK-293T human embryonic kidney cells using the Lentiviral Packaging Mix. A549/DDP cells were transfected with the lentivirus, and cells which highly expressed Meg3 (A549/DDP-Meg3 cells) were identified by selection with G418. The Meg3 sequence (NR_002766.2) was subcloned into an HIV type-1 lentiviral vector, pLV-GFP (Addgene, Cambridge, MA, USA) by restriction digestion using BamHI and MluI (New England Biolabs, Ltd., Hertfordshire, United Kingdom). The resulting constructs was termed pLV-Meg3 and pLV-GFP was used as a control. Following trypsinization, the cells were centrifuged at 1000 × g for 5 min and the supernatant was removed. A total of 1×10⁶ cells were subsequently seeded into six-well plates at high density (80–90% confluent). Lipofectamine 2000 (Invitrogen Life Technologies) complexes were prepared, according to the standard Lipofectamine protocol. Lipofectamine 2000 reagent (10 µl) and 100 pmol siRNA were each diluted in 250 µl Opti-MEM medium. Each mix was pooled and incubated for 5 min prior to the DNA-reagent complex being added to the cells (500 µl per well). The cells were incubated for 6 h at 37°C and subsequently incubated with DMEM, containing 10% FBS for 24 h.

The A549 cells were seeded at 50–80% cell density into six-well plates and transfected with 50 nM siRNAs specifically targeting Meg3 (siRNA/Meg3-243: 5′-GAUCCCACCAACAUACAAATT-3′; siRNA/Meg3-405: 5′-GCUCAUACUUUGACUCUAUTT-3′; siRNA/Meg3-852: 5′-CCCUCUUGCUUGUCUUACUTT-3′) (Shanghai Gene Pharma Co., Ltd., Shanghai, China) using Lipofectamine^®2000 (Invitrogen Life Technologies), according to the manufacturer's instructions, and were subsequently incubated for 24 h at 37°C in a 5% CO₂ incubator. The control siRNA sequence was as follows: 5′-UUAAGACGGUUGAAACUAG-3′.

Total RNA was extracted from the cells and blood samples using TRIzol^® (Invitrogen Life Technologies) and was subsequently reverse transcribed into cDNA using a reverse transcriptase kit (Takara Bio, Inc., Otsu, Japan). The relative mRNA expression levels of Meg3 were examined by RT-qPCR using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd., Dalian, China), and normalized to GAPDH. The RT-qPCR was performed using the ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR conditions were as follows: 94°C for 2 min, 94°C for 30 sec, 60°C for 30 sec and 72°C for 1 min for 30 cycles, and 72°C for 10 min. The PCR primer sequences used were as follows: MEG3, forward: 5′-ATCATCCGTCCACCTCCTTGTCTTC-3′ and reverse: 5′-GTATGAGCATAGCAAAGGTCAGGGC-3′; GAPDH, forward: 5′-AATGCCTCCTGCACCACCAAC-3′ and reverse: 5′-AAGGCCATGCCAGTGAGCTTC-3′. Calculations of expression were made using the 2^−ΔΔCT method.

Apoptosis of the cultured A549 and A549/DDP cells was evaluated using Annexin V labeling. An Annexin V-Allophycocyanin-Labeled Apoptosis Detection kit (Abcam, Cambridge, UK) was used according to the manufacturer's instructions. Staining with annexin V/PI was performed using an Annexin V-APC Apoptosis detection kit (Invitrogen Life Technologies). Briefly, an aliquot containing 2×10⁶ cells was resuspended in 0.5 ml binding buffer, labelled with 1 µl annexin V-APC plus 5 µl PI and were incubated for 10 min in the dark. The samples were immediately analyzed. Annexin negative and PI negative cells were designated as live cells and annexin positive and PI negative as early apoptotic cells. Staining with PI was performed using cell cycle detection kit (invitrogene, USA). Briefly, the cells were centrifuged at 500 × g for 10 min at room temperature and the supernatant was removed. An aliquot of ~1×10⁶ was incubated in ethyl alcohol at −20°C for 24 h and was subsequently incubated in 50 µg/ml PI in the dark, at room temperature for 30 min. The cells undergoing cell-cycle analysis were stained with propidium iodide, using the BD Cycletest Plus DNA Reagent kit (BD Biosciences, Shanghai, China), according to the manufacturer's instructions. The results were analyzed using a FACScan flow cytometer (BD Biosciences). The percentage of cells in each of the subG₀/G₁, G₁, S or G₂/M phases were counted and compared. Each experiment was performed at least three times.

The chemosensitivity of the cisplatin-resistant A549/DPP and parental A549 cells to cisplatin was determined by MTT assay. Briefly, the cells were seeded into 96-well plates (3.5×10³ cells/well) and treated with various concentrations of cisplatin (0, 1, 5, 8, 10, 15, 18, 20, 22 and 24 µg/ml). At 0, 24, 48, 72 and 96 h post-cisplatin application, cell survival was assessed using 0.5 mg/ml MTT (Sigma-Aldrich, St. Louis, MO, USA) solution. Following a 4 h incubation at 37°C, the medium was replaced with 150 µl dimethyl sulfoxide (Sigma-Aldrich) and vortexed for 10 min. The absorbance of each well at 490 nm was measured using a microplate reader 550 (Bio-Rad, Richmond, CA, USA). Each experiment was performed at least three times.

The A549/DDP cells were washed in phosphate-buffered saline (PBS) and lysed in radioimmuno-precipitation assay lysis buffer supplemented with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The total protein was quantified using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts (30 µg) of the whole cell lysates were separated by 5% SDS-PAGE (Beyotime Institute of Biotechnology, Jiangsu, China) and transferred onto polyvinylidene difluoride membranes (Merck Millipore GmbH, Eschborn, Germany). The blots were blocked in bovine serum albumin (5% w/v in PBS+0.1% Tween 20) for 1 h at room temperature and immunostained with the following antibodies at 4°C overnight: Mouse anti-human p53 (1:2,000; monoclonal, cat. no. ab26), mouse anti-human β-catenin (1:1,000; monoclonal, cat. no. ab6301), mouse anti-human survivin (1:500; monoclonal, cat. no. ab118046) and mouse anti-human GAPDH (1:5,000; monoclonal, cat. no. ab9484). These antibodies were purchased from Abcam (Cambridge, MA, USA). The membranes were washed four times with PBS, containing 0.1% Tween 20. The secondary antibody, goat anti-mouse (1:50,000; polyclonal, cat. no. ab97040; Abcam), was added in PBS, containing 0.1% Tween 20 for 1 h at 37°C. The membranes were then washed three times for 15 min with PBS, containing 0.1% Tween 20 (Sigma-Aldrich). The results were visualized using an enhanced chemiluminescent detection system (Pierce ECL Substrate Western Blot Detection system; Thermo Fisher Scientific, Waltham, MA, USA) and using a Molecular Imager ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analysis was performed using STATA 11 (StataCorp LP, College Station, TX, USA), and presented using GraphPad Prism 4.0 software (GraphPad Software, Inc., La Jolla, CA, USA). The results obtained from the in vitro assays are presented as the mean ± standard error of the mean from five separate experiments performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

A549/DDP is a cisplatin-resistant lung cancer cell line. The present study conducted an MTT assay to determine the half maximal inhibitory concentration (IC₅₀) of cisplatin in the A549/DDP and parental A549 cell lines. The IC₅₀ of cisplatin in the A549 and A549/CDDP cells was 5.68±2.54 µg/ml and 29.24±1.62 µg/ml, respectively (P<0.05; Fig. 1A). The A549/DDP cells exhibited a 5.15-fold greater resistance to cisplatin compared with that of the parental A549 cell line. To further investigate whether Meg3 had a significant role in the acquired cisplatin resistance of lung cancer cells, the expression levels of Meg3 were detected in the A549/DDP and parental A549 cells by RT-qPCR. The expression levels of Meg3 were significantly lower in the A549/DDP cisplatin-resistant cell line, as compared with in the A549 parental cell line (P<0.05; Fig. 1B).

The results above demonstrated that the expression levels of Meg3 were upregulated in A549 cells. The present study aimed to determine the expression levels of Meg3 in response to various concentrations of cisplatin in A549 cells. The cells were treated with various concentrations of cisplatin (0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 µg/ml) for 24 h. The relative expression levels of Meg3 were decreased in response to treatment with increasing concentrations of cisplatin (Fig. 1C). These results suggested that a reduction in Meg3 expression levels in the A549 lung cancer cell line was induced in response to cisplatin treatment.

To further evaluate the effects of Meg3 on the development of cisplatin resistance in lung cancer cells, a lentivirus overexpressing Meg3 was stably transfected into the A549/DDP cells (A549/DDP-Meg3). The transfection efficiency was validated by RT-qPCR. The expression levels of Meg3 in the A549/DDP-Meg3 cells were significantly upregulated, compared with those of the A549/DDP control cells (P<0.05; Fig. 1D). Furthermore, overexpression of Meg3 led to a reduction in the IC₅₀ of cisplatin in A549/DDP cells by ~9.79-fold (P<0.05; Fig. 1E).

In the A549/DDP-Meg3 cells treated with cisplatin (0.0, 1.0 and 1.5 µg/ml), upregulation of Meg3 resulted in an increased rate of cisplatin-induced apoptosis (P<0.05; Fig. 2A). Furthermore, the percentage of A549/DDP-Meg3 cells in the subG₀/G₁ and G₁ phases, particularly at checkpoint subG₀/_1, of the cell cycle gradually increased with the increasing doses of cisplatin (P<0.05; Fig. 2B). These results suggested that upregulation of Meg3 may reverse the cisplatin resistance of A549/DDP cells by inducing apoptosis and cell cycle arrest.

A549 cells were transfected with siRNA/control, siRNA/Meg3-243, siRNA/Meg3-405 or siRNA/Meg3-852, in order to analyze the role of Meg3 in determining the sensitivity of lung cancer cells to cisplatin. At 48 h post-transfection, the expression levels of Meg3 were significantly decreased by ~75.5% in the A549-siRNA/Meg3-852 cells compared with the siRNA/control-transfected cells (P<0.01; Fig. 3A). The Meg3 inhibition efficiency was higher with this siRNA than that of siRNA/Meg3-243 (30.1%) and siRNA/Meg3-405 (35.3%). Based on these results, siRNA/Meg3-852 was used to suppress the expression of Meg3 in A549 cells (Fig. 3B). The effects of Meg3 expression on the IC₅₀ of cisplatin in A549 cells were subsequently investigated. An MTT assay demonstrated that siRNA/Meg3-852 increased the IC₅₀ of cisplatin in A549 cells (P<0.05; Fig. 3C). Furthermore, A549-siRNA/Meg3-852 cells were treated with various concentrations of cisplatin (0.0, 1.0 and 2.0 µg/ml). An apoptosis assay demonstrated that knockdown of Meg3 by RNA interference reduced the rate of cisplatin-induced apoptosis in lung cancer cells (P<0.05; Fig. 4A). In addition, the percentage of siRNA/Meg3-transfected A549 cells in subG₀/G₁ and G₁ phases, particularly in checkpoint G₁, of the cell cycle increased with increasing doses of cisplatin (P<0.05; Fig. 4B).

A previous study reported that Meg3 was able to regulate the specificity of p53 transcriptional activation (17). The tumor suppressor gene p53 has a central role in tumor suppression, and mediates the functions of numerous tumor suppressors, in particular those associated with apoptosis and drug resistance of cancer cells (18). Furthermore, it has been demonstrated that the overexpression of wild-type p53, by transfection or DNA damage, downregulates the expression of β-catenin in human cells (19). The inhibitory effects of p53 on β-catenin have been revealed to be mediated by the ubiquitin-proteasome system and require activation of glycogen synthase kinase-3β (GSK-3β) (19). WNTs regulate embryonic development and determine cell fate, and dysregulation of WNT/β-catenin signaling may induce the development and/or progression of various human diseases, including cancer (20). Therefore, analyzing the effects of potential therapies on the WNT/β-catenin signaling pathway may represent a novel method for the screening of putative anti-cancer drugs. Gao et al (21) identified potential drugs that were able to inhibit WNT/β-catenin signaling, including cisplatin, using a drug screening platform based on β-catenin/transcription factor-mediated transcriptional activity. In addition, cisplatin was shown to activate WNT/β-catenin signaling in A549/DDP cells, and inactivate it in A549 cells (21). Based on these prior studies, the present study hypothesized that Meg3 may regulate the cisplatin resistance of cancer cells via the inhibition of β-catenin/survivin by activated p53. To examine this hypothesis, the protein expression levels of p53, β-catenin and survivin were determined in A549/DDP-Meg3 and A549/DDP cells by western blot analysis. Upregulation of p53 and downregulation of β-catenin/survivin was observed in the A549/DDP-Meg3 cells, and conversely the expression levels of these proteins were reversed in the A549/DDP cells (Fig. 5A). These findings suggested that upregulation of Meg3 in A549/DDP cells may decrease cisplatin resistance via inhibition of WNT/β-catenin signaling.

In a clinical setting, cisplatin is frequently used to treat patients with advanced lung cancer. In the present study, blood samples (n=27) were collected from patients with advanced lung cancer in order to determine the effects of cisplatin treatment on Meg3 expression. RT-qPCR analysis demonstrated that the expression levels of Meg3 were decreased in patients with advanced lung cancer following treatment with cisplatin-based chemotherapy, compared with those prior to cisplatin-based chemotherapy (P=0.0068; Fig. 5B). These results suggested that Meg3 may have a significant role as a therapeutic target in lung cancer.