A total of 25 NSCLC tissues and the adjacent normal lung tissues were obtained from patients (n=25; 13 male and 12 female; aged 39–78 years) admitted to Tianjin Huanhu Hospital (Tianjin, China) between March 2013 and March 2016. All of the samples were obtained with the patients' informed consent. The entire investigation conformed to the principles outlined in The Declaration of Helsinki. The present study was approved by the ethical review committees of Tianjin Huanhu Hospital.

Human NSCLC cell lines, including H650, A549, H522, H1299 and H1155 were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and human normal bronchial epithelial cells (HBECs) were purchased from Shanghai Maisha Biotechnology (http://maishabio.biogo.net/; Shanghai, China). The cells were routinely grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin/streptomycin mix (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 2 mM glutamine (Gibco; Thermo Fisher Scientific, Inc.), 5 mM glucose (Sigma Aldrich; Merck KGaA) and 1 mM sodium pyruvate (Sigma Aldrich; Merck KGaA) at 37°C in a humidified atmosphere containing 5% CO₂.

Total RNA was extracted from cultured cells and NSCLC tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. miRNAs from cancer specimens or cells were extracted using an RNeasy kit or miRNeasy mini kit (Qiagen GmbH, Hilden, Germany), respectively, according to the manufacturer's protocols. miRNAs and mRNAs were reverse transcribed using a miScript reverse transcription kit (Qiagen GmbH) following the manufacturer's protocols. qPCR was performed using a miRNA-specific TaqMan MiRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols using a Applied Biosystems 7500 Fast Real-Time PCR system (Thermo Fisher Scientific, Inc.). The qPCR conditions were as follows: 94°C pre-denaturation for 5 min, followed by 33 cycles of denaturation at 94°C for 30 sec, annealing and synthesis at 58°C for 30 sec. Relative gene expression data was analyzed using the 2^−ΔΔCq method (17). The primers used for RT-qPCR were as follows: miR-577-RT 5′-GTCGTATCCAGTGCAGGGTCCGAGGTGCACTGGATACGACCAGGTA-3′; oligod T 5′-TTTTTTTTTTTTTTTTTT-3′; U6-RT 5′-GTCGTATCCAGTGCAGGGTCCGAGGTGCACTGGATACGACAAAATATGG-3′; miR-577-qPCR, forward 5′-TGCGGTAGATAAAATATTGG-3′, reverse 5′-GTGCAGGGTCCGAGGT-3′; U6-qPCR, forward 5′-GCTTCGGCAGCACATATACTAAAAT-3′, reverse 5′-CGCTTCACGAATTTGCGTGTCAT-3′; WNT2B-qPCR, forward 5′-GCTGGACCAAACCTGAAC-3′, reverse 5′-CAAGAAGTATCGGGAAGC-3′; and β-actin-qPCR, forward 5′-CCGTCTTCCCCTCCATCGTGGG-3′, reverse 5′-CGCAGCTCATTGTAGAAGGTGTGG-3′.

WNT2B was overexpressed using PCR-amplified cDNA of H522 cells, which was cloned between the KpnI and XbaI restriction sites into the pcDNA3 vector (Beyotime Institute of Biotechnology, Shanghai, China). Overexpression was confirmed by RT-qPCR and western blot analysis. The pcDNA3 vector alone was used as the control group. In order to overexpress miR-577, the primary miR-577 was amplified from genomic DNA of H522 cells and cloned into the pcDNA3 vector between the BamHI and EcoRI restriction sites. To inhibit the function of miR-577, the 2-O-methyl-modifed antisense oligonucleotide of miR-577 (ASO-miR-577) and the scramble control oligonucleotides (ASO-NC) from the Shanghai GenePharma, Co., Ltd. (Shanghai, China) were used. The pSilencer/shR-WNT2B (shR-WNT2B) plasmid expressing siRNA targeting WNT2B was constructed by annealing double-stranded hairpin cDNA and inserting it into the pSilencer 2.1-U6 neo vector (Ambion; Thermo Fisher Scientific, Inc.) at the BamHI and EcoRI sites. The primers and sequences used are listed as follows: Pri-miR-577-sense (S) 5′-CGGGGTACCTGGTAGGTGCCCTGTTGA-3′, pri-miR-577-antisense (AS) 5′-CCGGAATTCTGGAAAGTAACCACGAGA-3′; ASO-miR-577 5′-CAGGUACCAAUAUUUUAUCUA-3′; ASO-NC 5′-UCACAACCUCCUAGAAAGAGUAGA-3′; WNT2B-S 5′-CGGGGTACCGCCACCATGTTGGATGGCCTTGGAGTG-3′; and WNT2B-AS 5′-TGCTCTAGATCAGGTTTGGTCCAGCCACTCTGCC-3′; shR-WNT2B-S 5′-GATCCCGGACTGATCTTGTCTACTTTCTCGAGAAAGTAGACAAGATCAGTCCGTTTTTGA-3′; shR-WNT2B-AS 5′-AGCTTCCGGACTGATCTTGTCTACTTTCTCGAGAAAGTAGACAAGATCAGTCCGG-3′. For transfection, H522 and A549 cells were seeded in 12-well plates at a density of 1×10⁷ cells/ml for 24 h, and were subsequently transfected with 2 µg plasmids using Lipofectamine^® 2000 (DNA:Lipofectamine^® 2000=1:2; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols and incubated for 4 h at 37°C. Subsequently, the cells were cultured in DMEM supplemented with 10% FBS for 48 h at 37°C prior to further analysis.

The hypothetical target of miR-577 was predicted using miRDB (http://www.mirdb.org/) (18), miRNA.org and TargetScan human7.1 (http://www.targetscan.org/vert_71/), which revealed that the 3′UTR of WNT2B may be complementarily paired with the seed sequences of miR-577.

The WNT2B 3′UTR was cloned into a pcDNA3/EGFP vector (Shanghai GeneChem Co., Ltd., Shanghai, China) and mutations were introduced at potential miR-577 binding sites. To determine that the 3′UTR of WNT2B mRNA is directly targeted by miR-577, 2×10⁶ H522 and A549 cells were cotransfected with 0.5 µg pri-miR-577 or 20 nM ASO-miR-577 and 0.5 µg 3′UTR of WNT2B or the mutant 3′UTR of WNT2Bin in 48-well plates using Lipofectamine^® 2000 (DNA:Lipofectamine^® 2000=1:2; Invitrogen; Thermo Fisher Scientific, Inc.). The binding site of miR-577 in the WNT2B 3′UTR was mutated as follows: 5′ UAACAUUAUUAACAUUUAGAA3′. After 48 h, the EGFP activity was measured using a spectrophotometer set at 528 nm. Red fluorescent protein-expressing plasmid was integrated as a transfection efficiency control.

H522 and A549 cells transfected as aforementioned were seeded in 96-well plates at a density of 5×10³ cells/well. A Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to detect the viability of H522 and A549 cells according to the manufacturer's instructions. The absorbance at 450 nm was measured.

For the colony formation ability assay, H522 and A549 cells were counted at 24 h post-transfection and seeded into 24-well plates at 500 cell/well. Culture medium was replaced every 3 days. After ~2 weeks, cells were washed with 1XPBS. Subsequently, colonies were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 1% crystal violet at room temperature for 20 min. The number of colonies was counted under an inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany).

H522 and A549 cells transfected with the indicated plasmids were collected and suspended in serum-free medium. Subsequently, 6×10⁵ cells were added to the upper chamber covered with 12.5 mg Matrigel (BD Biosciences) in 50 ml PBS, the lower chamber was filled with medium containing 20% FBS. Following incubation at 37°C for 48 h, cells below the membrane were fixed and stained with 0.5% crystal violet at room temperature for 30 min, washed with 1X PBS, air-dried and observed under an inverted microscope (Olympus Corporation, Tokyo, Japan). Cell invasion was assessed using a Transwell system (Corning Incorporated, Tewksbury, MA, USA). The number of migrated and invaded cells was counted under a microscope.

A total of 6×10⁸ A549 cells were lysed using the Protein Extraction kit according to the manufacturer's protocols (http://www.wanleibio.cn/; Wanlei Biotechnology, Beijing, China); 30 µg total proteins were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing Tween-20 for ~2 hat room temperature, prior to incubation with the primary antibodies. The membranes were probed with anti-E-cadherin (1:1,500; cat. no. ab1416; Abcam, Cambridge, MA, USA), anti-intercellular adhesion molecule 1 (ICAM1; 1:3,000; cat. no. ab223659; Abcam), anti-Vimentin (1:2,000; cat. no. ab188499; Abcam), anti-WNT2B (1:2,000; cat. no. ab50575; Abcam), anti-β-catenin (1:5,000; cat. no. ab16051; Abcam), anti-cyclin D1 (1:5,000; cat. no. ab15196; Abcam), anti-c-Myc (1:5,000; cat. no. ab39688; Abcam), p-GSK3β (1:1,000; cat. no. ab131097; Abcam), total-GSK3β (1:3,000; cat. no. ab2602; Abcam) and anti-GAPDH (1:5,000; cat. no. ab9485; Abcam) antibodies overnight at 4°C. Subsequently, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. 8889; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at 37°C. An enhanced chemiluminescence system (Thermo Fisher Scientific, Inc.) was used to detect the immunoreactive bands. The relative protein expression levels were normalized to that of GAPDH. The protein expression levels were measured using Image Pro Plus software v.6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

To determine the transcriptional activity of the Wnt pathway, 6×10⁶ A549 and H522 cells treated as indicated were co-transfected with either the Wnt signaling reporter TOP Flash or the negative control FOP Flash (EMD Millipore) according to the manufacturer's protocols. A549 and H522 cells were transiently transfected with either 2 µg pTOP flash or pFOP flash plasmids and 0.5 µg pSV40-Renilla plasmid as an internal control (Promega Corporation, Madison, WI, USA) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 48 h. The dual luciferase reporter assay system (Dual-Luciferase^® Reporter Assay system; cat. no. E1910; Promega Corporation) was used to assay the firefly and Renilla luciferase activity ratio.

A549 cells transfected with specific plasmids were seeded in 24-well plates at the indicated time points before immunofluorescence staining. The cells were washed in PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After cells were washed with PBS, the cells were permeabilized using 0.25% Triton-X-100 for 5 min at room temperature and blocked in 10% donkey serum (BeyotimeInstitute of Biotechnology; Nanjing, China) for 30 min. The cells were subsequently incubated with primary antibodies against β-catenin (1:100; cat. no. ab16051; Abcam) overnight at 4°C. The following day, cells were washed in PBS and then incubated at room temperature for 1 h with a fluorescent-labeled secondary antibody (1:200; cat. no. A0562; BeyotimeInstitute of Biotechnology), followed by incubation with DAPI (1:1,000; cat. no. C1002; BeyotimeInstitute of Biotechnology). Images were captured under a confocal microscope.

All analyses were performed using SPSS v. 19.0 for Windows (IBM Corp., Armonk, NY, USA) and GraphPad Prism v. 5.0 for Windows (GraphPad Software Inc., La Jolla, CA, USA). For comparisons of two treatment groups, a Student's t-test was used. For comparisons of three or more groups, one-way analysis of variance was followed by the Bonferroni post hoc test for comparison of two selected treatment groups; the Dunnett's post hoc test was used for comparisons of the other treatment groups with the corresponding controls. The Pearson's correlation analysis was used to determine the r-value. Associations between miR-577 expression and clinicopathological characteristics were assessed using chi-squared test. Data from at least three independent experiments are presented as the means ± standard deviation, or medians with ranges. P<0.05 was considered to indicate a statistically significant difference.

The relative expression levels of miR-577 in NSCLC tissues and cells were measured via RT-qPCR. The results revealed that miR-577 expression levels were significantly lower in NSCLC tissues than that in the adjacent normal tissues (Fig. 1A). In addition, the miR-577 expression levels in NSCLC patients were inversely associated with the tumor size, TNM stage and lymph node metastasis, but not the histological grade, age and gender (Table I). Furthermore, the expression levels of miR-577 were also detected in various human NSCLC cells lines including H650, A549, H522, H1299 and H1155 cells. As presented in Fig. 1B, miR-577 expression levels were lower in NSCLC cell lines compared with HBECs (Fig. 1B). These results suggested that miR-577 may have a suppressive role in NSCLC progression.

To further confirm whether miR-577 may affect NSCLC tumorigenesis, pcDNA3, pri-miR-577, ASO-NC or ASO-miR-577 was transfected into H522 and A549 cells. The results of RT-qPCR analysis demonstrated that the expression plasmids were effective (Fig. 1C). CCK-8 and colony formation assays revealed that the overexpression of miR-577 significantly inhibited cell proliferation and knockdown of miR-577 significantly promoted cell proliferation of H522 and A549 cells (Fig. 1D and E). Transwell migration and invasion assays revealed that ectopic expression of miR-577 significantly reduced the cell migration and invasion ability of H522 and A549 cells; knockdown of miR-577 increased H522 and A549 cells migration and invasion (Fig. 1F and G). These suggest that miR-577 may have a suppressive effect on NSCLC metastasis. In order to investigate the regulation of EMT by miR-577, the expression levels of the EMT markers, E-cadherin, Vimentin and ICAM-1 were analyzed by western blotting in A549 cells and the results indicated that the expression levels of Vimentin and ICAM-1 were significantly decreased by miR-577 overexpression but increased by miR-577 knockdown compared to the control groups; in addition, the expression levels of E-cadherin were significantly increased by miR-577 overexpression, but attenuated by miR-577 knockdown (Fig. 1H).

To investigate the mechanism of miR-577 affecting the biological behavior in NSCLC cells, the target genes of miR-577 were predicted using miRDB, miRNA.org and TargetScan human7.1; WNT2B was predicted to be a target of miR-577 (Fig. 2A). To verify that miR-577 can directly target WNT2B mRNA, EGFP reporter plasmids containing the 3′UTR or the 3′UTR-mut of WNT2B were constructed. In H522 and A549 cells, the relative EGFP level was significantly reduced in the pri-miR-577 and wild type 3′UTR of WNT2B co-transfected group. Additionally, co-transfection with ASO-miR-577 and wild type 3′UTR of WNT2B significantly increased the relative EGFP activity (Fig. 2B); however, no significant differences in EGFP activity were observed when H522 and A549 cells were co-transfected with pri-miR-577 or ASO-miR-577 and mutational EGFP reporter plasmids (Fig. 2C). To further confirm the regulation of WNT2B by miR-577, RT-qPCR and western blot assays were performed. The results revealed that overexpression of miR-577 significantly decreased the mRNA and protein levels of WNT2B; miR-577 knockdown significantly increased the expression of WNT2B at the mRNA and protein levels, suggesting that miR-577 downregulated WNT2B expression at the mRNA and protein levels in H522 and A549 cells (Fig. 2D and E). In addition, the present study reported that the mRNA levels of WNT2B in tumor tissues were negatively correlated with the miR-577 levels using Pearson's correlation analysis (r=−0.8743, R²=0.7644; Fig. 2F). These data indicated that WNT2B may be negatively regulated by miR-577 in NSCLC cells, which is the novel target of miR-577.

To determine the role of WNT2B in the aggressiveness of NSCLC, the mRNA expression levels of WNT2B in NSCLC cell lines were analyzed by RT-qPCR. The results demonstrated that WNT2B expression levels were significantly upregulated in NSCLC cells compared with in HBECs (Fig. 3A). In addition, the protein expression levels of WNT2B were detected by western blot assays, which also revealed that WNT2B was increased in NSCLC cells compared with in HBECs cells (Fig. 3B). As presented in Fig. 3C and D, the overexpression or knockdown of WNT2B were effective in H522 and A549 cells. At 24, 48 and 72 h post-transfection, cell viabilities of H522 and A549 cells were significantly increased by WNT2B overexpression and decreased by WNT2B knockdown (Fig. 3E). In addition, the relative colony formation rate was significantly increased by pWNT2B-transfected and decreased by shR-WNT2B-transfected compared with in the control groups (Fig. 3F). To identify whether WNT2B also influences the migration and invasion of NSCLC cells, Transwell migration and invasion assays were performed in H522 and A549 cells. As expected, overexpression of WNT2B significantly promoted cell migration and invasion ability (Fig. 3G and H). Finally, whether WNT2B may regulate the protein expression levels of the EMT process was investigated, including E-cadherin, ICAM-1 and Vimentin. Western blot analysis demonstrated that the overexpression of WNT2B significantly decreased E-cadherin expression, but increased ICAM-1 and Vimentin expression levels in A549 cells (Fig. 3I). Collectively, these results indicated that upregulation of WNT2B promotes NSCLC malignant behavior.

To address the miR-577 whether regulates the Wnt/β-catenin signaling pathway in NSCLC cells, the expression levels of WNT-associated genes and the activity of β-catenin were analyzed in the context of miR-577 overexpression or miR-577 overexpression in conjunction with pWNT2B in NSCLC cells. The nuclear distribution of β-catenin in A549 cells was decreased in pri-miR-577-transfected cells and increased in pWNT2B-transfected cells (Fig. 4A). Western blot assays revealed that the expression levels of phosphorylated (p)-GSK3β, activated β-catenin, c-Myc and cyclin D1 were significantly reduced by miR-577 overexpression and significantly increased by WNT2B overexpression (Fig. 4B). In addition, a TOP/FOP flash assay was performed, which is often used to detect Wnt/β-catenin signaling. The results demonstrated that miR-577 overexpression decreased the TOP/FOP flash ratio, but WNT2B overexpression increased it in NSCLC cells, indicating inhibition and activation of the Wnt/β-catenin signaling pathway, respectively (Fig. 4C).