Human gastric carcinoma cell lines, MGC-803 and HGC-27, were purchased from the China Center for Type Culture Collection (Wuhan, China). MGC-803 and HGC-27 cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA). To expose the cells to hypoxia, the cells were cultured in a Billups-Rotenburg chamber (Billups-Rothenberg, Inc., Del Mar, CA, USA) with 94% N₂, 1% O₂ and 5% CO₂ at 37°C for a certain time.

miR-18a mimics and negative control were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The cells were plated to 50% confluency and transfected with 200 nM miR-18a mimics or negative control using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. At 24 or 48 h after transfection, the cells were harvested for the further experiments.

At 48 h after transfection, the cells were harvested and 500-µl samples were added to fluorescence-activated cell sorting tubes. The solutions were mixed with 25 ng/ml fluorescein isothiocyanate-labeled annexin V and 10 mg/ml propidium iodide, and incubated for 15 min at room temperature in the dark. Subsequently, the cells were analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA).

MGC-803 and HGC-27 cells were transfected with the miR-18a-5p mimics, HIF-1α siRNA or negative control, cultivated for 24 h and transferred to the top of Matrigel-coated chambers (24-well insert; 8-µm pore size; BD Biosciences) in serum-free RPMI 1640 medium. Medium containing 20% fetal calf serum was added to the lower chamber as a chemoattractant. Following incubation for 24 h, the non-invaded cells were removed from the upper well with cotton swabs, while the invaded cells were fixed with 4% paraformaldehyde, stained with hematoxylin, and photographed (magnification, x200) in five independent fields for each well, using an optical microscope (Olympus Corporation, Tokyo, Japan). Each test was performed in triplicate.

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer's instructions. For cDNA synthesis, 2 µg total RNA was mixed with 500 ng oligo(dT) primers (Promega Corporation, Madison, WI, USA) or miRNA specific primers. The samples were incubated at 65°C for 10 min with 5 µl 5X first-strand buffer, 2 µl dNTP (5 mM), 20 units RNasin (Takara Bio, Inc., Otsu, Japan), 1 µl M-MLV reverse transcriptase (Promega Corporation) and distilled water to a total volume of 25 µl. The quantitative PCR assay mixture contained 12.5 µl 2X SYBR Green PCR Mix (Fermentas; Thermo Fisher Scientific), 0.3 µM gene-specific forward and reverse primers, and 1 µl cDNA template, which was made up to a final volume of 25 µl with distilled water. Thermal cycling parameters were set as follows: Initial activation at 95°C for 5 min, followed by 35 amplification cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 15 sec. The levels of gene expression were calculated by relative quantification against GAPDH or U6 snRNA, which were used as the internal controls, with U6 snRNA used as an internal reference gene for the qPCR detection of MiR-18a. All samples were amplified in triplicate, and the data analysis was conducted using MxPro qPCR system software (Stratagene, La Jolla, CA, USA).

MGC-803 and HGC-27 cells (2×10⁶) were collected and washed twice with ice-cold phosphate-buffered saline (PBS). The cell pellets were suspended in radioimmunoprecipitation assay lysis buffer for 30 min on ice, after which the lysates were centrifuged at 12,000 × g at 4°C for 20 min. Equal amounts of protein in the supernatant were isolated by 12% SDS polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA, USA). The membranes were blocked for 1 h at 37°C with 5% non-fat milk, and subsequently incubated with mouse monoclonal anti-HIF-1α (1:500; #359702, BioLegend, Inc., San Diego, CA, USA), anti-Bax (1:1,000; Abcam, Cambridge, MA, USA), Bcl-2 (1:1,500; #ab117115, Abcam), caspase 3 (1:1,000; #ab2171, Abcam), caspase 9 (1:1,000; #ab28131, Abcam) and GAPDH (1:400; #sc-365062, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibodies in 5% non-fat milk for 1 h at 37°C. After washing in PBS with 0.5% Tween 20 (PBST), the membrane was incubated with a rabbit anti-mouse horseradish peroxidase-conjugated secondary antibody (1:4,000; #ab6728, Abcam) at room temperature for 1 h. After further washing with PBST, the membrane was assayed with enhanced chemiluminescence with a chemiluminescence HRP subtrate (Millipore Corporation) and visualized on X-ray films.

To construct a luciferase reporter vector, the wild-type 3′-UTR or the mutant 3′-UTR of HIF-1α, which contained putative binding sites for miR-18a-5p, were subcloned into the psiCHECK-2 vector (Promega Corporation). MGC-803 cells were plated at a density of 5×10⁴ cells per well in 24-well plates. The following day, psiCHECK-2 luciferase vectors, which included the 3′-UTR of HIF-1α and miR-18a-5p mimics or negative control oligonucleotides, were transfected into the cells using Lipofectamine 2000. At 48 h after transfection, the luciferase assays were performed using a dual luciferase reporter assay system (Promega Corporation).

Statistical analysis was performed using the SPSS 19.0 software package (IBM SPSS, Armonk, NY, USA). All numerical data were analyzed using the Student's t-test, and all the tests performed were two-sided. P<0.05 was considered to indicate a statistically significant difference.

To investigate the function of miR-18a in gastric carcinoma cells under hypoxic conditions, the expression level of miR-18a in MGC-803 and HGC-27 cell lines was initially detected. As shown in Fig. 1, the expression level of miR-18a was downregulated in the two cell lines subjected to hypoxic conditions. In the HGC-27 cells, the miR-18a expression level under hypoxic conditions was 89% lower compared with the expression under normoxic conditions. Furthermore, in the MGC-803 cell line, the miR-18a expression level under hypoxic conditions was 64% lower compared with the expression under normoxic conditions.

The apoptosis rate of the gastric carcinoma cells was investigated using flow cytometry following subjection to hypoxic conditions for 24 h. As shown in Fig. 2A, miR-18a markedly affected the rate of apoptosis in the HGC-27 and MGC-803 cell lines. Early and late apoptosis rates (lower right and upper right quadrants of the FITC graphs, respectively; Fig. 2A) for the HGC-27 cells were 5.8 and 4.9%, respectively, transfected with negative control (NC) mimics. In addition, early and late apoptosis rates for the MGC-803 cells were 9.6 and 2.9%, respectively, transfected with NC mimics. By contrast, in the cells overexpressing miR-18a subjected to hypoxic conditions, the early and late apoptosis rates of HGC-27 cells were 23.9 and 8.0%, respectively. In addition, the early and late apoptosis rates of the MGC-803 cells were 29.3 and 13.6%, respectively, in the cells subjected to hypoxic conditions and miR-18a overexpression. As shown in Fig. 2B, the total apoptosis rates of HGC-27 and MGC-803 cells increased 2.98-fold and 3.43-fold following miR-18a mimics transfection, respectively.

The invasion ability of gastric carcinoma cells was investigated using a Transwell assay following the induction of hypoxic conditions for 24 h. As shown in Fig. 3A, miR-18a markedly affected the invasion ability of the HGC-27 and MGC-803 cells. The numbers of HGC-27 cells that had invaded the chamber following transfection with the negative control mimics or the miR-18a mimics were 152±8 and 49±5, respectively. Furthermore, the numbers of invasive MGC-803 cells following transfection with the negative control or miR-18a mimics were 165±7 and 42±4, respectively. As shown in Fig. 3B, the total number of invasive cells in the HGC-27 and MGC-803 cell lines decreased by 66.76 and 74.55% following miR-18a mimics transfection, respectively.

To understand the mechanisms through which miR-18a induces cell apoptosis and inhibits tumor invasion, several computational methods were used to identify miR-18a targets in humans, and the predicted duplex formation between the 3′-UTR of HIF-1α and miR-18a is shown in Fig. 4A. Among the ~275 targets predicted using the TargetScan 6.2 software program, HIF-1α had been previously implicated in the regulation of cell apoptosis and/or invasion, which was of particular interest since the expression of HIF-1α may be induced under hypoxic conditions. To investigate whether HIF-1α was a direct target of miR-18a, a luciferase reporter assay was conducted. The results revealed that miR-18a inhibited the activity of the firefly luciferase with the wild-type 3′-UTR of HIF-1α. By contrast, miR-18a exhibited no effect on the activity of the firefly luciferase with mutant type 3′-UTR of HIF-1α (Fig. 4B). Subsequently, the effect of miR-18a overexpression on the mRNA and protein expression levels of HIF-1α were investigated. Although miR-18a overexpression did not result in the degradation of HIF-1α mRNA (Fig. 4C), miR-18a overexpression did reduce the activity of the luciferase reporter gene fused to the wild-type HIF-1α 3′-UTR, indicating that miR-18a targets HIF-1α through translational inhibition. In accordance with these results, a clear reduction in the level of endogenous HIF-1α protein expression was observed in the MGC-803 and HGC-27 cells overexpressing miR-18 (Fig. 4D).

To investigate the possible mechanisms underlying the effects of miR-18a on cell apoptosis, the implication of miR-18a in a typical signaling pathway of apoptosis was evaluated. Thus, the protein expression levels of Bcl-2, Bax, caspase 3 and caspase 9 were detected in cells subjected to hypoxic conditions following transfection with the miR-18a mimics or negative control mimics. As shown in Fig. 5, Bcl-2 protein expression levels were downregulated following the induction of miR-18a overexpression, while Bax protein expression levels were upregulated in the MGC-803 and HGC-27 cells. In addition, two important genes in the apoptosis signaling pathway were analyzed. The protein expression levels of caspase 3 and caspase 9 were demonstrated to be upregulated following the induction of miR-18a overexpression. These results indicated that the apoptosis signaling pathway may be activated by miR-18a under hypoxic conditions.