Between July 2017 and February 2018, 32 samples of GC and adjacent tissues (5 cm away from tumor tissue) were obtained from patients, including 19 male and 13 female subjects, and the median age was 57 years (age range, 35–84 years). All patients underwent gastrectomy at the Changhai Hospital of Naval Medical University (Shanghai, China). Tissue samples from the patients with GC were immediately flash frozen in liquid nitrogen following resection at −196°C. The Changhai Hospital Ethics Committee (Shanghai, China) approved the present study and all patients provided written informed consent.

AGS and MKN45 human GC cells and GES human normal gastric cells were purchased from the American Type Culture Collection. AGS, MKN45 and GES-1 cells were cultured in RPMI-1640 medium (Hyclone; GE Healthcare Life Sciences) containing 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences), 100 IU/ml penicillin and 100 µg/ml streptomycin.

miR-502-5p mimic and miR-502-5p-mimic negative control (NC) was purchased from Shanghai GenePharma Co., Ltd. The microRNA was transfected into GC cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. The time interval between transfection and subsequent experimentation was 48 h.

Total RNA was extracted from the patient tissue samples, and AGS, MKN45 and GES cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific Inc.). miR-502-5p expression levels were measured using a TaqMan microRNA assay kit (Takara Bio. Inc.) according to the manufacturer's instructions, using U6 as an internal control. Total RNA was then reverse transcribed into cDNA using a Prime Script RT reagent kit (Takara Bio. Inc.) according to the manufacturer's instructions. SYBR-Green (Takara Bio. Inc.) was used to determine SP1 mRNA expression relative to β-actin. The thermocycling conditions for qPCR were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 10 sec, 60°C for 30 sec. The primers were designed as follows: miR-502-5p forward, 5′-CGGGCATCCTTGCTATCTG-3′ and reverse, 5′-CAGCCACAAAAGAGCACAAT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; SP1 forward, 5′-TGGCAGCAGTACCAATGGC-3′ and reverse, 5′-CCAGGTAGTCCTGTCAGAACTT-3′; and β-actin forward, 5′-CCTGGCACCCAGCACAAT-3′ and reverse: 5′-GGGCGGGACTCGTCATAC-3′. Each sample was analyzed in triplicate and levels were quantified using the 2^−ΔΔCq method (15).

AGS and MKN45 cells were transfected with miR-502-5p mimics or NC for 24 h as aforementioned. AGS and MKN45 cells were collected and 5×10³ cells per well were seeded into 96-well plates in triplicate. Following 1–4 days in the incubator at 37°C with an atmosphere of 5% CO₂, 10 µl MTT assay solution was added to each well for 4 h at 37°C. Next, 100 µl DMSO was added to each well for 30 min to dissolve the purple formazan, and optical density was measured at 490 nm with a microplate reader (Bio-Rad Laboratories, Inc.).

In the migration assay, 1×10⁵ cells were plated in 200 µl serum-free medium in the top Transwell chamber. In the invasive assay, 1×10⁵ cells were plated in 200 µl serum-free medium in the top Transwell chamber with a Matrigel-coated membrane. The matrigel was pre-coated at 37°C for 30 min. In both the migration and invasion experiments, 500 µl medium containing 10% FBS was added into the lower chamber as a chemoattractant. After 24 h, the cells on the top surface of the Transwell chamber were removed using cotton swabs. The cells on the bottom surface were fixed at room temperature with 100% methanol for 30 min, and then stained with 0.05% crystal violet for 30 min at room temperature. Five visual fields were randomly selected to photograph with an Olympus IX51 light microscope (Olympus Corporation; magnification, ×20).

Transfected AGS and MKN45 cells were lysed with RIPA buffer (Cell Signaling Technology, Inc.) containing complete protease inhibitor cocktail (Roche Diagnostics), phosphatase inhibitors (Roche Diagnostics), 5 mM dithiothreitol (DTT, Sigma-Aldrich; Merck KGaA) and 1 mM phenyl methyl sulfonyl fluoride (Sigma-Aldrich; Merck KGaA). The supernatant of the cell lysate was collected and protein concentrations were determined using the bicinchoninic protein assay kit (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Then 20 µg protein was loaded onto a 10% gel, resolved using SDS-PAGE and transferred onto PVDF membranes. Membranes were then blocked with 5% fat-free milk for 2 h at room temperature. Subsequently, membranes were incubated with primary antibodies against SP1 (1:1,000; cat. no. WL02251; Wanleibio Co., Ltd.) and β-actin (1:1,000; cat. no. P30002M; Abmart Pharmaceutical Technology Co., Ltd.) at 4°C overnight, washed three times with TBST (0.05% Tween-20) and incubated with anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. 7054S; Cell Signaling Technology, Inc.) at room temperature for 2 h. Following three washes with TBST, immunoreactive bands were visualized using ECL working fluid (Biochannel, Nanjing, China; http: //www.biochannel.cn/page19.html?product_id=299). This experiment was repeated three times.

Target Scan version.7.2 (http: //www.targetscan.org/), miRandaversion.2010 (http: //www.microrna.org/microrna/getGeneForm.do) and miRBase version.22.1 (http: //www.mirbase.org/) were used to predict the candidate target genes of miR-502-5p.

All data are expressed as the mean ± standard deviation, and statistical analyses were performed using SPSS version 17.0 (SPSS, Inc.). Student's paired t-tests were used for comparisons between two groups and one-way ANOVA followed by Tukey's post hoc test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Expression levels of miR-502-5p in 32 GC tissues and two GC cell lines were analyzed using RT-qPCR. The expression level of miR-502-5p in GC tissues was significantly lower compared with those in matched normal adjacent tissues (P<0.05; Fig. 1A). Compared with normal GES-1 gastric epithelial cells, the two GC cell lines exhibited significantly lower miR-502-5p expression (P<0.001; Fig. 1B).

To study the effect of miR-502-5p on GC cells, NC or mimics of miR-502-5p were transfected into MKN45 and AGSGC cells. The transfection efficiency of miR-502-5p was evaluated using RT-qPCR. The level of miR-502-5p in MKN45 and AGS cells transfected with miR-502-5p mimics was significantly higher compared with those transfected with NC (both P<0.001; Fig. 2A). Overexpression of miR-502-5p significantly decreased proliferation at all time points compared with the NC group (all P<0.01; Fig. 2B) and reduced the cellular migration and invasion capacities of MKN45 and AGSGC cells (Fig. 2C and D).

To investigate the molecular mechanism underlying miR-502-5p-mediated inhibition of GC progression, target genes of miR-502-5p were predicted using miRNA prediction software and databases. Target Scan 7.2, miR and a 2010 and miR Base 22.1 predicted that SP1 is a target gene of miR-502-5p. The mRNA level of SP1 decreased significantly in AGS and MKN45 cells transfected with miR-502-5p mimic (P<0.05 and P<0.01, respectively, Fig. 3A). In addition, western blotting demonstrated that the protein level of SP1 was lower in AGS and MKN45 cells transfected with miR-502-5p mimics compared with the controls. SP1 levels were also evaluated in GC cells. Compared with GES cells, the expression of miR-502-5p in AGS and MKN45 cells was lower (Fig. 1B), but the expression of SP1 was higher (Fig. 3C). The mRNA expression level of SP1 in four sets of tumor tissues was significantly higher compared with that in normal paracancerous tissues (Fig. 3D). This suggests that SP1 is targeted by miR-502-5p in GC.