Paired NSCLC and adjacent non-cancerous lung tissues were obtained from 115 patients during curative resection of NSCLC in Zhongnan Hospital of Wuhan University (Wuhan, China) between January 2010 and December 2013. These tissues were flash-frozen in liquid nitrogen immediately following resection and stored at −80°C prior to use. None of the patients had received neoadjuvant chemotherapy or radiotherapy prior to surgery. Patient characteristics are presented in Table I. The present study was approved by the Research Ethics Committee of Zhongnan Hospital of Wuhan University, and written informed consent was obtained from each patient.

A total of four NSCLC cell lines (A549, H460, 95D and HCC827) and normal lung epithelial cells (NLEC) were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin G sodium, and 100 µg/ml streptomycin sulfate (Sigma-Aldrich Shanghai Trading Co, Ltd., Shanghai, China). All the cells were incubated at 37°C in a humidified atmosphere with 5% CO₂.

For RNA transfection, 10⁵ cells were seeded into each well of 24-well plate and incubated overnight. Subsequently, the cells were transfected with mature miR-215 mimics, miR-215 inhibitors (anti-miR-215), or negative control (miR-NC or anti-miR-NC) (Shanghai GenePharma Co, Ltd., Shanghai, China) at a concentration of 50 nM using Lipofectamine® 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA).

The total RNA was extracted from the cells and tissues using TRIzol reagent (Invitrogen Life Technologies). Complementary (c)DNA was reverse transcribed from the total RNA samples using specific miR primers from the TaqMan MicroRNA assay and reagents from the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems Life Technologies, Foster City, CA, USA). The primers for miR-215 and U6 were as follows: miR-215 forward, 5′-GGGTCCGAGGTATTCGCACT-3′; miR-215 reverse, 5′-CGATGACCTATGAATTGACAGACG-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′; U6 reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′. Products were amplified by PCR using the TaqMan Universal PCR Master Mix kit (Applied Biosystems Life Technologies) and the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s and 74°C for 5 s. Small nucleolar RNA U6 was used as an internal standard for normalization. All the reactions were performed in triplicate, and the 2^−∆Ct method (∆CT = CT_miR-215 − CT_U6) was used to quantify the relative quantity of miR-215.

The in vitro cell proliferation was measured using the MTT method. Briefly, cells were seeded into 96-well plates (2×10⁴ cells/well) and incubated at 37°C following transfection. At various time-points (24, 48, 72 or 96 h), the culture medium was removed and replaced with fresh medium containing 0.5 mg/ml MTT (Sigma-Aldrich, St. Louis, MO, USA). The cells were then incubated for a further 4 h and resolved by dimethyl sulfoxide (Sigma-Aldrich). The absorbance was measured at 490 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

Apoptosis was detected by flow cytometric analysis. Briefly, the cells were washed and resuspended at a concentration of 1×10⁶ cells/ml. The cells were then stained with Annexin V and propidium iodide, using an Annexin V apoptosis detection kit (Abcam, Shanghai, China). Following incubation at room temperature in the dark for 15 min, cell apoptosis was analyzed with a FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA).

The invasion assay was performed using 24-well Transwell chambers (8 µm; Corning Life Sciences, Corning, NY, USA). Following transfection, tumor cells were resuspended in serum-free RPMI 1640 medium and 2×10⁵ cells were seeded into the upper chambers covered with 1 mg/ml Matrigel (BD Biosciences, San Jose, CA, USA), while 0.5 ml RPMI 1640 containing 10% FBS was added to the bottom chambers. Following a 24-h incubation, the non-filtered cells were gently removed with a cotton swab. Filtered cells located on the lower side of the chamber were stained with 0.1% crystal violet (Sigma-Aldrich) and counted under a microscope (DP50; Olympus Corporation, Tokyo, Japan).

The scratch migration assay was performed to evaluate the effect of miR-215 on NSCLC cell migration. When the cells transfected with miR-215 mimics, miR-215 inhibitors or NC reached confluence, a scratch in the cell monolayer was made with a cell scratch spatula. Following incubation of the cells under standard conditions for 24 h, images of the scratches were captured using a digital camera system coupled with a microscope (DP50; Olympus Corporation).

In order to identify potential mRNA targets of miR-215, database searches of microRNA target prediction engine TargetScan (http:www.targetscan.org) were conducted using the search term ‘miR-215’. The ZEB2 target was subsequently selected for further investigation as ZEB2 has been identified as an important oncogene in NSCLC (23) and a direct target of miR-215 in renal cell carcinoma (16).

The pGL3-report luciferase vector (Sigma-Aldrich Shanghai Trading Co, Ltd.) was used for the construction of the pGL3-ZEB2 and pGL3-ZEB2-mut vectors. The pGL3-ZEB2-mut vector was constructed using ZEB2 that had undergone site-directed mutagenesis of the miR-215 target site using the Quik-Change site-directed mutagenesis kit (Agilent Technologies GmbH, Waldbronn, Germany). For the luciferase reporter assay, the cells were cultured in 24-well plates (10⁵ cells/well) and transfected with the plasmids (100 ng/well) and miR-215 mimics using Lipofectamine 2000 (50 nM). At 24 h following transfection at 37°C, luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA). The firefly luciferase activity was normalized to the Renilla luciferase activity for each transfected well.

Protein lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Kangcheng Biology Engineering Co, Ltd., Shanghai, China). Following blocking in 5% non-fat milk in 1X Tris-buffered saline (pH 7.4) containing 0.05% Tween-20, the membranes were incubated with purified rabbit anti-ZEB2 antisera (cat. no. LS-C160768; dilution, 1:1,000; LifeSpan BioSciences, Inc., Seattle, WA, USA) at 4°C overnight. The following day, the membranes were washed with PBS and incubated with peroxidase-conjugated goat anti-rabbit IgG (cat. no. sc-2445; dilution, 1:4,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Immunodetection was conducted using chemiluminescence reagents (Pierce Biotechnology, Inc., Rockford, IL, USA) and exposed on X-ray film (Nikon Corporation, Tokyo, Japan). β-actin (cat. no. bs-0061R; Bioss, Inc., Woburn, MA, USA) was used as an internal reference for relative quantification.

Tumor formation was studied by establishing a xenograft model. Commercial lentiviral vectors containing miR-215 (LV-miR-215; Shanghai GeneChem Co. Ltd., Shanghai, China) were used to infect NSCLC cells according to the manufacturer's instructions. An empty lentiviral construct served as a negative control (LV-NC). The stably transfected cells were selected using puromycin (1.5 µg/ml; Kangcheng Biology Engineering Co, Ltd.). A total of 16 female BALB/c athymic nude mice (3–4 weeks old) were purchased from the Model Animal Research Center of Nanjing University (Jiangsu, China). NSCLC cells (1×10⁶; 100 µl cell suspension) stably overexpressing miR-215 or NC were inoculated subcutaneously into the mice (n=8 per group). Bidimensional tumor measurements were taken with vernier calipers every 4 days, and the tumor volume (mm³) was calculated using the formula volume = (length × width²)/2. Three weeks following inoculation, the mice were sacrificed by spinal dislocation and the tumors were weighed.

All data are presented as the mean ± standard deviation. Statistical analyses were performed using SPSS software, version 15.0 (SPSS Inc., Chicago, IL, USA). The differences between the groups were analyzed using Student's t-test or χ² test. The associations between miR-215 expression and ZEB2 protein levels were evaluated using Pearson's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR analysis was performed to detect miR-215 expression in NSCLC tissues and cell lines. As presented in Fig. 1A, the results demonstrated that the expression levels of miR-215 were significantly reduced in NSCLC specimens (8.2±1.9) compared with those in the corresponding adjacent non-cancerous tissues (19.2±4.0; P<0.001). The miR-215 expression in the 4 NSCLC cell lines was also markedly downregulated, compared with that of the NLECs (Fig. 1B). Since among the 4 NSCLC cell lines the A549 cell line exhibited the lowest miR-215 expression, while 95D cells expressed relatively high levels of miR-215, these two cell lines were selected for miR-215 mimics or miR-215 inhibitor transfection and further analysis.

ZEB2 protein levels were detected by using western blot analysis. The results demonstrated that the ZEB2 protein expression levels in the tumor samples were increased compared with those of the adjacent normal tissues (P<0.001; Fig. 1C). The ZEB2 protein expression levels in the NSCLC cells were also increased compared with those of the NLEC cells (Fig. 1D). In addition, a significant inverse correlation (R=−0.4216; P=0.0002) was observed between ZEB2 and miR-215 protein expression levels in NSCLC tumor tissues (Fig. 1E).

The associations between miR-215 expression and various clinicopathological parameters of NSCLC tissues are presented in Table I. The patients were divided into a high miR-215 expression group and a low miR-215 expression group, using the median miR-215 expression value amongst all 115 NSCLC patients as a cut-off. As demonstrated in Table I, miR-215 expression was significantly reduced in samples with lymph node metastasis (P=0.002) and advanced TNM stage (P<0.001). No significant differences were observed between miR-215 expression and age, gender, smoking status, cell types, T stage or tumor differentiation.

To selectively overexpress or downregulate miR-215, mature miR-215 mimics or miR-215 inhibitors were transfected into A549 or 95D cells. RT-qPCR analysis confirmed enhanced miR-215 expression following miR-215 mimics transfection and reduced miR-215 expression following miR-215 inhibitors transfection (Fig. 2A). The results of the MTT assay demonstrated that cell proliferation was significantly impaired in A549 cells transfected with miR-215 mimics, while proliferation of 95D cells was enhanced following miR-215 inhibitors transfection, compared with that of the corresponding controls (Fig. 2B).

Flow cytometry was employed to determine the effect of miR-215 on cell apoptosis. The proportion of apoptotic A549 cells transfected with miR-215 mimics was significantly increased compared with that of the negative control group. In addition, downregulation of miR-215 reduced 95D cell apoptosis (Fig. 2C).

A Transwell invasion assay was performed to investigate whether miR-215 had a direct influence on NSCLC cell invasion. As demonstrated in Fig. 2D, upregulation of miR-215 inhibited the invasion of A549 cells. Conversely, transfection of 95D cells with miR-215 inhibitors promoted cell invasion ability. The scratch migration assay also confirmed the inhibitory effect of miR-215 on NSCLC cell migration (Fig. 2E).

Using the bioinformatics software TargetScan for target gene prediction, ZEB2 was identified as a potential target of miR-215. The predicted binding of miR-215 with the ZEB2 3′UTR is illustrated in Fig. 3A. To further confirm that ZEB2 is the direct target of miR-215 in NSCLC, miR-215 mimics were transfected into A549 cells, which significantly reduced ZEB2 protein expression levels in these cells (Fig. 3B). Subsequently, the pGL3-ZEB2 and pGL3-ZEB2-mut plasmids were created. The luciferase reporter assay demonstrated that transfection of miR-215 mimics induced a marked reduction in luciferase activity of pGL3-ZEB2 plasmid in A549 cells, without altering the luciferase activity of pGL3-ZEB2-mut (Fig. 3C). These data indicate that ZEB2 is a direct target of miR-215 in NSCLC.

To further evaluate the effects of miR-215 on tumor growth in vivo, A549 cells were engineered to stably overexpress miR-215 by lentiviral infection. These cells were injected subcutaneously into nude mice to form ectopic tumors. The cells transfected with negative lentiviral vector LV-NC were also inoculated. As indicated in Fig. 4A–C, the tumors formed from miR-215-overexpressing A549 cells were smaller and exhibited reduced tumor weights compared with those of the control tumors. RT-qPCR analysis of the tumor tissues confirmed elevated miR-215 expression levels in miR-215-overexpressing tumors (Fig. 4D)