The human gastric cancer cell lines SGC-7901 and BGC-823 were obtained from the China Center for Type Culture Collection (Wuhan, China). All cell lines were maintained in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) under normoxic or hypoxic conditions for 7 days. Subsequently, the cells were treated with 20 nM rapamycin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) for 72 h at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Rapamycin was used to downregulate the expression levels of HIF-1α (18). For hypoxic exposure, tumor cells were incubated in an hypoxic incubator (BINDER GmbH, Tuttlingen, Germany) containing 1% O₂, which was balanced by CO₂ and nitrogen.

The Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay was used to evaluate cell viability, according to the manufacturer's protocol. Cells were seeded onto 96-well plates at a density of 5×10³ cells/well and incubated for 24 h, and subsequently incubated in culture medium containing 20 nM rapamycin at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. CCK-8 solution (10 µl) was added to each well at 24, 48 and 72 h. The color intensity was evaluated using a microplate reader (Beijing Liuyi Biotechnology Co., Ltd., Beijing, China) at an absorbance wavelength of 450 nm. All experiments were performed in triplicate and repeated independently three times.

These assays were performed according to a previously described protocol (19). Briefly, Transwell units with 8.0-µm-pore polycarbonate filters (Corning Incorporated, Corning, NY, USA) were precoated with 50 µl of 1:5 diluted Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). A total volume of 200 µl of gastric cancer cell suspension, which contained ~1×10⁵ rapamycin-treated cells, was added to the upper compartment of the precoated units. The units were then transferred to the wells of the culture plate and incubated for 24 h at 37°C. Subsequently, the cells and Matrigel on the upper surface of the membrane were removed. Cells that had migrated to the underside were stained with 0.1% crystal violet for 15 min at room temperature, and cell numbers were determined using light microscopy. Five randomly selected fields were counted per insert.

The gastric cancer cell lines were maintained with medium containing 20 nM rapamycin under normoxic or hypoxic conditions for 72 h. Total RNA was then isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The corresponding complementary DNA (cDNA) was synthesized using the PrimeScript™ RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). RT-qPCR was performed using SYBR^® premix Ex Taq™ (Takara Biotechnology Co., Ltd.) and gene-specific primers in the Rotor-Gene 3000 system (Qiagen, Inc., Valencia, CA, USA). The PCR reaction mixtures contained 12.5 µl Premix Ex Taq™, 0.2 µM PCR primers, 0.4 µM SYBR Green I and 0.08 µM cDNA. All primers were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA), and their sequences and annealing temperatures are presented in Table I. GAPDH was used as the housekeeping gene for normalization of the mRNA expression levels. Fold changes in the expression levels for each mRNA were calculated using the 2^−∆∆Cq method (20).

Using the TRIzol^® reagent, total protein was extracted from the cells. The protein concentration was evaluated using a Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The proteins (50 mg/well) were separated by 12% SDS-PAGE and then electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) at room temperature and then probed overnight at 4°C using polyclonal antibodies against CD44 (ab51037; Abcam, Cambridge, UK) and HIF-1α (sc-10790; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a final dilution of 1:200 (w/v). The anti-GAPDH antibody (sc-25778, Santa Cruz Biotechnology, Inc.) was used overnight at 4°C at a final dilution of 1:600 (w/v). The membranes were then washed in PBS with Tween-20 and incubated with peroxidase-conjugated anti-rabbit immunoglobulin G [dilution, 1:3,000 (w/v); BA1054; Wuhan Boster Biological Technology, Ltd., Wuhan, China) (21) for 30 min at room temperature. The bands were visualized using an enhanced chemiluminescence kit (P0018; Beyotime Institute of Biotechnology, Haimen, China), and their optical densities were evaluated using a photo documentation and imaging system (BIO-ID VL, Conn, France).

All data were expressed as the mean ± standard deviation. Using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA), one-way analysis of variance and the Student's t-test were employed to analyze the data. P<0.05 was considered to indicate a statistically significant difference.

Compared with the normoxia control group (Fig. 1), hypoxia significantly increased the viability of SGC-7901 and BGC-823 cells after 72 h of incubation (P=0.001 and P=0.009, respectively). Hypoxia-induced increases in cell viability were inhibited by rapamycin treatment (P=0.001 and P=0.01, respectively).

To examine the effects of hypoxia and rapamycin pretreatment on the biological behavior of the cells, migration and invasion assays were performed. As presented in Fig. 2, significantly higher numbers of invasive cells were observed following hypoxia treatment. Hypoxia induced 1.3- and 1.9-fold increases in the number of invasive SGC-7901 and BGC-823 cells, respectively (P=0.001 and P=0.005, respectively). However, rapamycin pretreatment significantly decreased these invasive cell numbers by 29 and 24%, respectively (P=0.001 and P=0.008, respectively).

Hypoxia increased the mRNA expression levels of HIF-1α in SGC-7901 and BGC-823 cells (P=0.001 and P=0.001, respectively; Fig. 3). To downregulate the mRNA expression of HIF-1α, the two gastric cancer cell lines were treated with rapamycin. Under hypoxic conditions, treatment with rapamycin significantly reduced the mRNA expression levels of HIF-1α by 40 and 30% in SGC-7901 and BGC-823 cells, respectively (P=0.001 and P=0.001, respectively).

Whether hypoxia is able to modulate the expression levels of CD44 mRNA was subsequently examined. Hypoxia induced 3.0- and 2.2-fold increases in CD44 mRNA expression levels in SGC-7901 and BGC-823 cells, respectively (P=0.01 and P=0.001, respectively; Fig. 4). Treatment with rapamycin under hypoxic conditions decreased CD44 mRNA expression levels by 45 and 52% in SGC-7901 and BGC-823 cells, respectively (P=0.007 and P=0.03, respectively; Fig. 4).

The protein expression levels of HIF-1α and CD44 in SGC-7901 and BGC-823 cells that were treated with or without rapamycin under hypoxic conditions were also examined. HIF-1α and CD44 protein expression levels were significantly induced by hypoxia in these two cell lines (SGC-7901 cells, P=0.001 and P=0.03, respectively; and BGC-823 cells, P=0.029 and P=0.032, respectively; Fig. 5). However, these increases in HIF-1α and CD44 protein expression levels were significantly reduced upon rapamycin treatment (SGC-7901 cells, P=0.009 and P=0.01, respectively; and BGC-823 cells, P=0.02 and P=0.037, respectively; Fig. 5).