The present study was approved by the Ethics Committee of Henan University and informed consent was obtained from all patients. Sixty-eight lung samples (lung adenocarcinoma and adjacent non-tumor lung tissues) were obtained from The First Affiliated Hospital of Zhengzhou University (Zhengzhou, China), and the Cancer Hospital of Henan Province (Zhengzhou, China) between November 2013 and March 2015. All the samples were frozen in liquid nitrogen immediately after resection until use. None of the patients enrolled in the study had received chemotherapy or radiotherapy. All tumors were histopathologically confirmed as lung adenocarcinoma by two independent qualified clinical pathologists. The clinical data of 68 cases of lung adenocarcinoma are shown in Table I.

Total RNA was extracted from lung adenocarcinoma and adjacent non-tumor tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA (1 µg) was used to synthesize cDNA. The expression level of miR-125a-5p was determined using a High-Specificity miR-125a-5p qRT-PCR Detection kit (Stratagene Corp., La Jolla, CA, USA) using qRT-PCR (ABI 7500 Fast System; Applied Biosystems, Foster City, CA, USA). All protocols were performed according to the manufacturer's instructions. U6 small nuclear RNA (U6 snRNA) was used as an endogenous control for normalization. Relative expression level of miR-125a-5p was calculated using the 2^−ΔΔCt method. miR-125a-5p primers were as follows: RT-primer, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGGACTC-3′; forward, 5′-TCCGAAGTGTCCAATTTCCC-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; U6 primers were as follows: RT-primer, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGA CAAAATATGGAACTGC-3′; forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′.

Human lung adenocarcinoma cell lines (A549 and H1299) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Both cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS; Gibco-BRL, Gaithersburg, MD, USA) and incubated at 37°C/5% CO₂.

The miR-125a-5p mimic (GMR-miR™ miRNA-125a-5p mimic) and the scrambled oligonucleotide used in the present study were synthesized by Shanghai GenePharma Co. Ltd. (Shanghai, China). For cell transfection, lung adenocarcinoma cell lines A549 and H1299 were seeded into 6-well plates at a density of 1.5×10⁵ cells/well. Once cells reached 50~80% confluency, transient transfection was conducted using Lipofectamine™ 2000 (Invitrogen), and the working concentration of miR-125a-5p mimic was 50 nM. Cells from each cell line were subdivided into three groups: the blank group (blank) that was non-transfected, scramble group (scramble) that was transfected with the scrambled oligonucleotide, and the miR-125a-5p mimic group (miR-125a-5p mimic) that was transfected with the miR-125a-5p mimic.

Cells (blank, scramble and miR-125a-5p mimic) were seeded into a 96-well plate at a density of 1×10⁴ cells/well, with five replicate wells/group. The absorbance value for each well was determined using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). The optical density (OD) was detected using CCK-8 reagents after 0, 24, 48 and 72 h of cultivation. The OD was recorded using a microplate reader (ELx800; BioTek, Winooski, VT, USA), at a wavelength of 450 nm (OD₄₅₀). The experiments were independently triplicated.

Transwell filters (Costar, Cambridge, MA, USA) were coated with Matrigel (3.9 µg/µl, 60–80 µl) on the upper surface of a polycarbonic membrane (6.5-mm diameter, 8-µm pore size). After 30 min of incubation at 37°C, the Matrigel solidified and served as the extracellular matrix for tumor cell invasion analysis. Three groups of cells (blank, scramble and miR-125a-5p mimic) were adjusted to 2×10⁵ cells/ml, and resuspended in 200 µl of serum-free medium in the upper chambers, and the lower chamber was supplied with 500 µl of medium containing 10% FBS, followed by incubation at 37°C in 5% CO₂ for 48 h. After incubation, the medium was removed from the upper chamber and cells in the upper chamber were carefully removed with a cotton swab, and then stained with Methylene Blue Staining solution (Beyotime, Haimen, China). The number of cells invading the Matrigel was counted from three randomly selected visual fields, using an inverted microscope at a magnification of ×200. All experiments were performed in triplicate.

Three groups of cells (blank, scramble and miR-125a-5p mimic) were harvested at 48 h post-transfection by trypsinization, and were adjusted to 1×10⁶ cells/ml in 1X binding buffer. After staining with FITC-Annexin V and propidium iodide (PI) using the FITC-Annexin V Apoptosis Detection Kit I (BestBio, Shanghai, China), cells were analyzed using a FACScan^® flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Cells from each treatment group (blank, scramble and miR-125a-5p mimic) were harvested at 48 h post-transfection. Then caspase activity was conducted using a caspase activity assay kit (Beyotime) according to the manufacturers instructions. Cellular extracts and substrates (Ac-DEVD-pNA) were kept into 96-well plates for 2 h at 37°C. Absorbance values were recorded using a microplate reader (ELx800), at a wavelength of 405 nm.

The human NEDD9 3′ UTR fragments containing putative binding sites for miR-125a-5p were amplified by PCR from human genomic DNA. Overlap extension PCR was used to obtain the mutant NEDD9 3′ UTRs. These DNA fragments were cloned into a pmir-GLO reporter vector (Promega, Madison, WI, USA), downstream of the luciferase gene, to generate the recombinant vectors, wild-type-3′ UTR and mutant type 3′ UTR. For the luciferase reporter assay, A549 cells were transiently co-transfected with miRNA (miR-125a-5p mimic or scrambled-125a-5p) and reporter vectors (wild-type-3′ UTR or mutant type 3′ UTR), using Lipofectamine™ 2000. Luciferase activities were measured 48 h post-transfection using the Dual-Luciferase Assay kit (Promega), according to the manufacturer's instructions.

Total proteins of the cultured cells were extracted using RIPA buffer containing phenylmethanesulfonyl fluoride (PMSF) 48 h post-transfection. The protein concentrations were determined using BCA protein assay kit (Beyotime). Proteins (30 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% BSA in 0.05% Tween-20-TBS for 1 h, the membranes were incubated overnight at 4°C with diluted (1:1,000) primary antibodies (polyclonal rabbit anti-NEDD9; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After extensive washing with TBST, the membranes were incubated with diluted (1:3,000) anti-rabbit IgG-HRP secondary antibody (Santa Cruz Biotechnology, Inc.). Signals were detected using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). GAPDH (Santa Cruz Biotechnology, Inc.) was used as an endogenous reference.

All analyses were performed using SPSS 17.0 software. All data are expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to analyze the multiple groups data. A value of P<0.05 was considered statistically significant.

The clinicopathological characteristics of the 68 lung adenocarcinoma cases included in the present study are presented in Table I. We found that the miR-125a-5p expression level in lung adenocarcinoma tissues was associated with differentiation status, tumor-node-metastasis (TNM) stage and lymph node metastasis (P<0.05; Table I). No significant differences were observed between miR-125a-5p expression and sex or age. Using adjacent non-tumor tissues as reference, miR-125a-5p expression in lung adenocarcinoma tissues was found to be significantly reduced (P<0.05; Fig. 1A). Compared to adjacent non-cancerous tissues, the expression level of NEDD9 in lung adenocarcinoma tissues was higher (P<0.05; Fig. 1B). To investigate the correlation between miR-125a-5p and NEDD9, we examined their expression levels in primary human lung adenocarcinoma tissues. Unlike the matched normal lung tissues, in the tumor tissues from the 68 patients with lung adenocarcinoma, miR-125a-5p was reduced, whereas NEDD9 protein was increased, which demonstrated a significant negative correlation (R²=0.632; P<0.01; Fig. 1C). The data suggest that the expression of NEDD9 and miR-125a-5p have an inverse correlation in lung adenocarcinoma tissues.

To test the proliferation effects of miR-125a-5p on lung adenocarcinoma cells, we performed CCK-8 assay. The corresponding cell growth curves are presented in Fig. 2. There were no significant differences in OD₄₅₀ values between the blank and scramble groups (P>0.05). However, compared to the blank and scramble groups, the OD₄₅₀ values for the miR-125a-5p mimic group on 24, 48 and 72 h were significantly decreased (P<0.05) in both the A549 (Fig. 2A) and H1299 (Fig. 2B) cell lines.

To test the effect of miR-125a-5p on the invasive ability of lung adenocarcinoma cells, we performed Transwell assay. We found that the mean number of cells that penetrated the Transwell membrane was significantly lower in the miR-125a-5p mimic group than these numbers in the blank and scramble groups (P<0.05) in both the A549 (Fig. 3B) and H1299 (Fig. 3C) cell lines.

Our FCM results indicated that the apoptosis level of cells transfected with the miR-125a-5p mimic was significantly enhanced compared to these levels in the cells in the blank and scramble groups (P<0.05) in both the A549 (Fig. 4A) and H1299 (Fig. 4B) cell lines. Similarly, transfection of the A549 and H1299 cells with the miR-125a-5p mimic was found to significantly increase caspase-3/−7 activity compared to cells in the blank and scramble groups (P<0.05) in both the A549 (Fig. 4C) and H1299 (Fig. 4D) cell lines.

Bioinformatic analyses using TargetScan (www.targetscan.org) and miRanda (www.microrna.org) predicted that the 3′ UTR of NEDD9 contains binding sites for miR-125a-5p (Fig. 5A). Subsequent western blot analysis indeed showed that NEDD9 expression was downregulated in the A549 and H1299 cells following transfection with the miR-125a-5p mimic (P<0.05; Fig. 5B). To verify whether NEDD9 is a direct target of miR-125a-5p, we used a dual-luciferase reporter system containing either wild-type or mutant 3′ UTR of NEDD9, respectively. Co-transfection with miR-125a-5p and the reporter vector containing the wild-type-3′ UTR significantly suppressed luciferase activity (P<0.05; Fig. 5C). These results indicate that miR-125a-5p negatively regulates NEDD9 expression by directly binding to putative binding sites in the 3′ UTR.

In order to verify whether miR-125a-5p functions by targeting NEDD9 in lung adenocarcinoma cells, we divided cells into four groups (blank, scramble, miR-125a-5p mimic and siRNA NEDD9), and conducted CCK-8 and FCM assays. Western blot analysis indeed showed that NEDD9 expression of the miR-125a-5p mimic and siRNA NEDD9 groups were downregulated (P<0.05) in the A549 (Fig. 6A) and H1299 cells (Fig. 6B). CCK-8 assay results showed that the OD₄₅₀ values of miR-125a-5p mimic and siRNA NEDD9 groups on days 24, 48 and 72 h were significantly decreased (P<0.05) in both the A549 (Fig. 6C) and H1299 (Fig. 6D) cells, compared to the blank and scramble groups. Results of FCM indicated that the apoptosis levels in the miR-125a-5p mimic and siRNA NEDD9 groups were significantly enhanced compared to those of the cells in the blank and scramble groups (P<0.05) in both the A549 (Fig. 6E) and H1299 (Fig. 6F) cell lines. These results indicate that miR-125a-5p suppresses proliferation and induces cell apoptosis by targeting NEDD9 in A549 and H1299 cells.