A total of 15 gastric cancer and adjacent non-cancerous tissue samples were obtained from patients that had undergone surgical treatment for gastric cancer at The First Affiliated Hospital of Zhengzhou University (Zhengzhou, China) between March 2011 and March 2013. The patients were diagnosed independently by two experienced pathologists from The First Affiliated Hospital of Xinxiang Medical University, according to the Cancer Staging Manual published by the American Joint Committee on Cancer. The present study was approved by the Ethics Committee of the Medical College of Zhengzhou University and informed consent was obtained from the patients prior to sample collection, conforming to the Declaration of Helsinki and the local legislation. Written informed consent was obtained from all patients prior to the start of the study.

Human gastric cancer MKN-45, SGC-7901 and MGC-803 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA), while the human gastric epithelial mucosa GES-1 cell line was purchased from the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (BioTeke Corporation, Beijing, China) supplemented with 10% heat-inactivated Invitrogen fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a humidified cell incubator with 5% CO₂ at 37°C.

A miR-524-5p mimic and inhibitor, alongside their corresponding negative controls (scramble miRNA), were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The SGC-7901 and MGC-803 cells were seeded in 6-well plates at 30% confluence one day prior to transfection. The cells were transfected with miR-524-5p mimic, miR-524-5p inhibitor and control miRNA using Invitrogen Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Total RNA was extracted from the cell lines and the tissue samples using Invitrogen TRIzol reagent (Thermo Fisher Scientific, Inc.) and DNAse (catalog no. ab32124; Abcam, Cambridge, MA, USA). RT was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. The expression of miR-524-5p was verified by stem-loop RT-qPCR with specific RT and PCR primers. The primer sequences (Sangon, Shanghai, China) were: Matrix metallopeptidase (MMP)-2, sense, 5′-CCCCAGACAGGTGATCTTGAC-3′ and antisense, 5′-GCTTGCGAGGGAAGAAGTTG-3′; and MMP-9, sense, 5′-CGCTGGGCTTAGATCATTCC-3′ and antisense, 5′-AGGTTGGATACATCACTGCATTAGG-3′. U6 small nuclear RNA was used as an internal control. RT-qPCR for MMP-2 and MMP-9 was performed using the following conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. qPCR was performed on an Applied Biosystems® 7500 thermocycler (Thermo Fisher Scientific, Inc.) using SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. The comparative CT method, ∆∆Ct, was used to quantify the data. Briefly, ΔCt was calculated by subtracting the CT of U6 or GAPDH mRNA from the mRNA of interest, and ΔΔCt was calculated by subtracting the ΔCt of the negative control from the ΔCt of the samples. The data was normalized according to the study conducted by Schmittgen and Livak (11).

Transfected cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed using a cell lysis buffer consisting of 50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP-40, 50 mM NaF and 1 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China). The cells were centrifuged at 10,000 × g for 15 min at 4°C. The protein concentration was measured by BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Cell protein lysates were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel with the Mini-PROTEAN® II Electrophoresis System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using Mini-Protean precast gels (Bio-Rad Laboratories, Inc.) at 200 V for 1 h, and subsequently transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were next blocked with 5% skimmed milk in Tris-buffered saline (pH 7.4) containing 0.05% Tween 20 (TBST), and incubated with primary antibodies, including anti-MMP-2 (mouse; monoclonal; dilution, 1:10; catalog no. ab80737; Abcam, Cambridge, UK), anti-MMP-9 (mouse; monoclonal; dilution, 1:25; catalog no. ab51203; Abcam) and anti-β-actin (mouse; monoclonal; dilution, 1:5000; catalog no. ab75373; Abcam) overnight at 4°C. The membranes were washed three times in TBST for 10 min, and subsequently incubated with HRP conjugated goat anti-rabbit IgG secondary antibodies (catalog no. MBS560261; Mybiosource, San Diego, CA, USA). Chemiluminescence detection was performed using Pierce™ Fast Western Blot kit (Thermo Fisher Scientific, Inc.) and ECL Substrate (GE Healthcare Life Sciences, Chalfont, UK). The densitometry analysis was analyzed using Image-Pro Plus software, version 1.61.

The transfected cells were seeded onto 96-well plates (Corning Incorporated, Corning, NY, USA) at a density of 1×10⁴ cells/well. In total, 20 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT; Sigma-Aldrich) at a concentration of 5 mg/ml was added to the transfected cells, and incubated at 37°C for 4 h. Following the removal of the culture medium, the remaining crystals were dissolved in dimethyl sulfoxide (Sigma-Aldrich), and the absorbance at 570 nm was measured. All experiments were performed in triplicate.

Cell apoptosis analyses were performed as previously described, with certain modifications (12). Briefly, SGC-7901 and MGC-803 cells were detached using trypsin (Sigma-Aldrich), washed twice in PBS, centrifuged at 1,000 × g for 5 min and resuspended in 195 µl Annexin V-fluorescein isothiocyanate (FITC) binding buffer (Sigma-Aldrich). Subsequently, 5 µl Annexin V-FITC (BD Biosciences, Erembodegem, Belgium) was added to the cells. Following an incubation period of 15 min in the dark at room temperature, 400 µl binding buffer was added. The percentage of apoptotic cells was analyzed using flow cytometry (FACSCalibur™; BD Biosciences, Franklin Lakes, NJ, USA) and CellQuest Pro™ Software (version 5.2.1; BD Biosciences).

Cell invasion was determined using 24-well Transwell chambers coated with Matrigel™ (BD Biosciences, Bedford, MD, USA) in order to determine the effect of miR-524-5p on gastric cancer cell invasion. Briefly, 8-µm pore size filters (Sigma-Aldrich) were coated with 100 µl Matrigel™ (1 mg/ml; dissolved in serum-free RPMI-1640). A total of 600 µl RPMI-1640 containing 10% Invitrogen FBS (Thermo Fisher Scientific) was added to the lower chamber, while single cell suspensions (1×10⁵ cells/well) were added to the upper chamber. Cells were incubated for 48 h at 37°C, and non-invading cells were removed using cotton swabs (Takara Biotechnology Co. Ltd.). Invaded cells were stained with 0.1% crystal violet (Takara Biotechnology Co., Ltd.) for 10 min at room temperature and examined using the Olympus BX51 fluorescence microscope (Olympus Optical Co., Tokyo, Japan).

The results are presented as the mean ± standard deviation. The Student's t-test was used to assess the statistical significance of differences between groups. The data were analyzed by SPSS version 11.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to determine the expression levels of miR-524-5p in gastric cancer, the present study evaluated the expression levels of miR-524-5p in human gastric cancer tissues and cell lines using RT-qPCR. As demonstrated by Fig. 1A, the expression levels of miR-524-5p were markedly lower in gastric cancer tissues, compared with normal gastric mucosa tissue. Similarly, the expression levels of miR-524-5p were significantly decreased in MKN-45, SGC-7901 and MGC-803 cells, compared with human normal gastric epithelial mucosa GES-1 cells (Fig. 1B). These results demonstrate that miR-524-5p is downregulated in gastric cancer tissues and cell lines. SGC-7901 and MGC-803 cells were selected for the subsequent in vitro experiments, since the mRNA expression levels of miR-524-5p were lower in these cells than in MKN-45 cells.

Since miR-524-5p was significantly downregulated in gastric cancer, the present study concluded that miR-524-5p may inhibit growth, promote apoptosis and enhance invasion in gastric cancer cells. To verify this hypothesis, the expression levels of miR-524-5p in SGC-7901 and MGC-803 cells were upregulated by transfecting the cells with miR-524-5p mimics, as detected by RT-qPCR (Fig. 2). Subsequently, an MTT assay was performed to evaluate the effects of miR-524-5p on the growth of SGC-7901 and MGC-803 cells. As demonstrated by Fig. 3, miR-524-5p overexpression inhibited the growth of SGC-7901 and MGC-803 cells, and the inhibitory effect of miR-524-5p was markedly increased with increasing transfection times.

Cell apoptosis was detected using propidium iodide and Annexin V-FITC staining following 48 h of miR-524-5p mimic transfection. The percentage of cells undergoing apoptosis was measured using flow cytometry. Following transfection with miR-524-5p mimics, the percentage of apoptosis significantly increased to 30.7% in SGC-7901 cells (Fig. 4A) and 38.8% in MGC-803 cells (Fig. 4B), compared with 10.1 and 9.8%, respectively, in the control groups. These results demonstrate that miR-524-5p is critical in promoting the apoptosis of gastric cancer cells.

The present study examined the effect of miR-524-5p on gastric cancer cell invasion. As demonstrated by Fig. 5, the number of invasive cells was significantly decreased in SGC-7901 and MGC-803 cells transfected with miR-524-5p mimics, compared with miR-control-transfected cells (SGC-7901 miR-524-5p-transfected vs. control cells, 85 vs. 173 cells; MGC-803 miR-524-5p-transfected vs. control cells, 139 vs. 222 cells). These results indicate that overexpression of miR-524-5p inhibits the invasion ability of gastric cancer cells in vitro.

Numerous studies have demonstrated that extracellular MMP-2 and MMP-9 are overexpressed in various types of cancer (13–15). As demonstrated in Fig. 6, the protein expression levels of MMP-2 and MMP-9 were significantly decreased in SGC-7901 (Fig. 6A) and MGC-803 (Fig. 6B) cells transfected with miR-524-5p mimics. Similarly to the results of western blot analysis, a decrease in the mRNA levels of MMP-2 and MMP-9 was observed using RT-qPCR (Fig. 6C and D). These results demonstrate that transfection with miR-524-5p attenuates gastric cancer cell invasion by inhibiting the expression of MMP-2 and MMP-9.