The gastric cancer cell lines SGC-7901, AGS, MKN-45, HGC-27 and BGC-823, and the normal gastric epithelial cell line GES-1, were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂.

Total RNA was extracted from the cell lines using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), followed by RT. qPCR was performed with an ABI 7500 system (Thermo Fisher Scientific, Inc.) using TaqMan Universal PCR Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). Thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 1 min. The expression of U6 small nuclear RNA was determined as an internal control. The primers used for RT-qPCR were as follows: miR-144: 5′-GCGCGAATTCGAGATCTTAACAGACCCTAGCTC-3′ (forward primer) and 5′-GCGCGGATCCGTGCCCTGGCAGTCAGTAGG-3′ (reverse primer); U6snRNA: 5′-CGCAAGGAUGACACGCAAAUUCGUGAAGCGUUCCAUAUUUUU-3′. Fold changes in relative gene expression were calculated using the 2^−ΔΔCq method (15). All reactions were performed in triplicate.

miR-144 mimic (5′-UACAGUAUAGAUAUGUACU-3′), miR-144 antisense (5′-AGUACAUCAUCUAUACUGUA-3′) and miR-144 scramble (negative control, 5′-AUCAUCUAUACUGUAAGUAC-3′) were obtained from Chang Jing Bio-Tech, Ltd. (Changsha, China). For transfection, HGC-27 cells were seeded in 6- or 24-well plates and transiently transfected at 70–80% confluence using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The final concentration of miR-144 mimic, miR-144 antisense or miR-144 scramble in the transfection system was 100 nM. After 24 h, the cells were used for the subsequent experiments.

HGC-27 cell proliferation was evaluated using an MTT assay. A total of 5×103 cells were seeded into each well of a 96-well plate and incubated for 24, 48 or 72 h. Subsequently, 20 µl MTT (0.5 mg/ml in DMSO) was added to each well. Following removal of the medium, absorbance was determined at 570 nm using a spectrophotometer.

The HGC-27 cells for apoptosis analysis were harvested, washed twice with ice-cold PBS, and resuspended in 1Xbinding buffer at a concentration of 6×105 cells/ml. A total of 100 µl of the solution (6×104 cells) was then transferred to a 5 ml culture tube. The cells were then incubated with 5 µl of Annexin V-fluorescein isothiocyanate (BD Biosciences, San Jose, CA, USA) and 5 µl of 1 mg/ml propidium iodide (PI) at room temperature in the dark for 15 min. Following the incubation period, 400 µl of 1X binding buffer was added to each sample and the samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences). Results were obtained by analyzing data with FlowJo software (version 7.6.1; FlowJo LCC, Ashland, OR, USA). All assays were repeated three times.

The migration and invasion of HGC-27 cells were assayed using Transwell chamber (Corning Inc., Corning, NY, USA). For the migration assays, 1×105 cells were plated in the top chamber containing a non-coated membrane. The cells were plated in the serum-free medium (Invitrogen; Thermo Fisher Scientific, Inc.), and medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS was used as a chemoattractant in the lower chamber. For invasion assays, the filters were precoated with Matrigel (BD Biosciences) in the upper chamber prior to cell seeding. Subsequently, 2×104 cells were seeded into the upper chamber and the lower chamber was filled with culture medium supplemented with 10% FBS. The cells were incubated at 37°C in a tissue culture incubator with 5% CO₂. After 16 h, cells that had migrated and invaded to the lower membrane surface were fixed with 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 10 min and stained with 0.1% crystal violet for 10 min at room temperature. Images were captured by a phase-contrast microscope (Zeiss AG, Oberkochen, Germany). Five random fields from each membrane were photographed and cells were counted. These experiments were performed in triplicate.

HGC-27 cells were harvested and lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris/HCl and 1% NP-40, pH 7.5). Protein concentration was determined using the Bradford assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts (50 µg) of proteins were separated by SDS-PAGE (8% gels) and then transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 10% non-fat milk in PBS for 1 h at room temperature, membranes were incubated overnight with anti-AP4 antibodies (cat. no. sc-166024; dilution, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies (cat. no. 7074; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. The signals were detected with the SuperSignal West Pico Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Protein levels were normalized to total GAPDH, using a monoclonal anti-GAPDH antibody (cat. no. sc-293335; dilution, 1:1,000; Santa Cruz Biotechnology).

Potential miR-144 targets were predicted and analyzed using the following two publicly available databases: TargetScan 6.2 (http://www.targetscan.org), and miRanda (http://www.microrna.org/microrna/getGeneForm.do) (16,17).

The 3′-untranslated region (3′-UTR) of the AP4 gene that was predicted to interact with miR-144 was amplified and inserted into pMIR-Report Luciferase vector (Ambion; Thermo Fisher Scientific, Inc.). Mutations within potential miR-144-binding sites were generated by nucleotide replacement. Co-transfection of indicated plasmids plus 1 ng pRL-TK Renilla was performed using Lipofectamine^® 2000 (Promega Corporation, Madison, WI, USA). After 24 h, the activities of firefly luciferase and Renilla luciferase in the cell lysates were measured with the Dual-Luciferase reporter assays (Promega Corporation, Madison, Wisconsin, USA), and values for cells with reporter genes containing the wild-type AP4 3′-UTR were set equally to 1. Experiments were performed three times.

AP-4 DNA sequences were amplified from human genomic DNA, subcloned into the pcDNA3.1+ vector (Invitrogen; Thermo Fisher Scientific, Inc.), and verified by DNA sequencing. miR-144 mimics/mimic control and the pc-AP4 plasmids were co-transfected into cultured HGC-27 cells by using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated for 48 h.

Data are presented as the mean ± standard deviation from at least three separate experiments. Statistical differences were analyzed using SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA). Comparisons between two groups were performed using Student's t-test. Comparisons among three or more groups were performed using one-way analysis of variance followed by a least significant difference post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To delineate the function of miR-144 in gastric epithelial cell malignant transformation, its expression levels were analyzed in GC cells and normal gastric epithelial cells. As presented in Fig. 1A, results from RT-qPCR analysis revealed that the expression level of miR-144 was significantly lower in the five GC cell lines SGC-7901, AGS, MKN-45, HGC-27 and BGC-823 compared with the normal gastric epithelial cell line GES-1. These data suggest that the expression of miR-144 is downregulated in GC.

To clarify the role of miR-144 in GC progression, a miR-144 mimic was transfected into HGC-27 cells, which had the lowest expression level of miR-144. The effect of miR-144 mimic was confirmed by RT-qPCR (Fig. 1B). miR-144 overexpression significantly suppressed cell proliferation as revealed using an MTT assay (Fig. 2A). In addition, the proportion of apoptotic cells significantly increased in miR-144-transfected cells (Fig. 2B and C). Therefore, the results suggest that upregulation of miR-144 may inhibit GC cell proliferation and promote apoptosis in vitro.

Subsequently, the role of miR-144 in regulating GC cell migration and invasion was investigated using cells transfected with miR-144 mimic or control. As demonstrated by Transwell and Matrigel assays, overexpression of miR-144 significantly inhibited the migratory and invasive capabilities of GC cells (Fig. 2D and E).

The aforementioned results suggested an inhibitory role for miR-144 in GC. Therefore, to investigate the target of miR-144 in GC and reveal the underlying molecular mechanisms, miR-144 targets were searched for using TargetScan. The conserved target gene AP4, which has been identified as a transcription factor, was selected for subsequent investigation (Fig. 3A). As presented in Fig. 3B, overexpression of miR-144 significantly inhibited AP4 expression, whereas inhibition of miR-144 resulted in an increased protein level of AP4, which suggests that AP4 is a target of miR-144. To further confirm that AP4 is a direct target of miR-144, a luciferase reporter assay was performed. Notably, miR-144 overexpression significantly suppressed the luciferase activity of the wild-type AP4 3′-UTR, which was fully rescued when the potential miR-144-binding site was mutated (Fig. 3C).

To further verify the functional association between miR-144 and AP4, the present study assessed whether ectopic expression of AP4 could reverse the inhibitory effects of miR-144 on GC cells. As presented in Fig. 4A, transfection with pc-AP4 could increase AP4 expression levels. The increased expression of AP4 significantly attenuated the tumor-suppressive effect of miR-144, underlining the importance of AP4 for miR-144 action in cell proliferation, migration and invasion (Fig. 4B-D).