The SMMC-7721 human HCC cell line was obtained from the Shanghai Institute of Cell Biology at the Chinese Academy of Science (Shanghai, China). The present study was approved by the Ethics committee of the Third Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Scientific Inc., Waltham, MA, USA). Cell cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂. When the cells covered 70–80% of the bottle bottom, they were digested using 0.25% trypsin (Gibco). The cells in the logarithmic phase of growth were used for the following experiments. For hyperthermia treatment, the cells were subjected to heat shock in an incubator (Forma Series II 3110; Thermo Fisher Scientific, Inc.) preheated to 40, 42 or 44°C for 1 h. A control group was maintained at 37°C. The temperature was monitored and maintained within 0.1°C during the treatment period. Following heat shock treatment, the cells were then re-incubated at 37°C for the indicated time periods.

Cells were seeded at an initial density of 1×103 cells/well in 96-well plates. Following 24 h culture at 37°C in a humidified atmosphere containing 5% CO₂, the cells were exposed to hyperthermic conditions (40, 42 or 44°C) separately for 1 h and recovered to 37°C. After a further 24 h, cell morphology was observed using an inverted microscope (TE2000-U; Nikon Corporation, Tokyo, Japan) and cell viability was measured using a Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. For cells exposed to 42°C, cell viability was additionally detected at 12, 24, 48, 72 and 96 h following hyperthermia. The optical absorbance at a wave length of 450 nm was measured using a microplate reader (Model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The percentage of growth inhibition was calculated using the following formula: 1 - (Absorbance hyperthermia/absorbance at 37°C) × 100%. The growth curves were constructed using average absorbance at 450 nm (optical density [OD], 450 nm) from three independent experiments.

The antisense plasmid pEGFP-C1-mTOR was donated by Dr Yu Guo (Liver Transplant Center, The Third Affiliated Hospital of Sun Yat-Sen University). Transient transfection was conducted using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. SMMC-7721 cells were seeded in 24-well plates (5×10⁴ cells/well) one day prior to transfection until they reached 80% confluence. A total of 1 µg plasmid DNA and 3 µl transfection reagent were used to transfect the cells in each well in the absence of serum. After 4 h, the medium was replaced with DMEM supplemented with 10% FBS. The expression of green fluorescent protein was observed using the TE2000-U inverted fluorescence microscope.

SMMC-7721 cells were seeded in 6-well plates (1×105 cells/well), and cells were collected 36 h post-transfection. Total RNA was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA was reverse transcribed and amplified using a one-step RT-PCR kit (Beijing TransGen Biotech Co., Ltd., Beijing, China). Genomic DNA was removed using phenol/chloroform extraction. Next, 1 µg isolated RNA was included in a 20-µl reaction mixture together with 0.5 µl Forward Primer, 0.5 µl Reverse Primer, 12 µl 2X One-Step Reaction Mix and 0.5 µl EasyScript One-Step Enzyme Mix. RNA was reverse transcribed and amplified using an ABI 2700 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA). The reaction was performed under the following conditions: 94°C for 12 min, and then 30 cycles 94°C for 30 sec, 54°C for 30 sec and 72°C for 30 sec followed by a final extension at 72°C for 5 min. The mRNA expression levels of mTOR were measured using the following gene-specific primers: mTOR forward, 5′-CGCTGTCATCCCTTTATCG-3′ and reverse, 5′-ATGCTCAAACACCTCCACC-3′. The relative mRNA expression levels of mTOR were normalized against those of β-actin using the following gene-specific primers: β-actin forward, 5′-GGACTTCGAGCAAGAGATGG-3′, and reverse, 5′-AGCACTGTGTTGGCGTACAG-3′. The primers were synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). The amplified PCR products were subjected to electrophoresis in 1% agarose gel (Sangon Biotech Co. Ltd., Shanghai, China), then stained with ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA). All experiments were repeated at least three times. The photo-density ratio of the RT-PCR product for mTOR and β-actin was used to identify the expression levels of the target gene. The optical density of each sample band was captured using a UV gel imaging system (GDS-8000; Ultra-Violet Products Ltd., Cambridge, UK) and analyzed using Quantity One Image software (Bio-Rad Laboratories, Inc.). The density ratio of the RT-PCR product for mTOR and β-actin was used to identify the expression levels of the target gene.

Post-transfection, cells were harvested, washed twice in ice-cold phosphate-buffered saline (PBS; Wuhan Boster Biological Technology Ltd., Wuhan, China) and then lysed on ice for 30 min in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, Shanghai, China). After centrifugation at 10,000 × g for 5 min, the supernatant was harvested to obtain the total cellular protein extract. The protein concentrations were assessed using a bicinchoninic acid protein assay kit (Beijing Comwin Biotech Co., Ltd., Beijing, China). After the protein concentrations were assessed, 50 µg total protein was separated using 10% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred onto nitrocellulose membranes (Beijing Comwin Biotech Co., Ltd.). Following blocking in 5% non-fat dry milk in 25 mM Tris-buffered saline (150 mM NaCl) with 0.1% Tween 20 buffer (Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., Shanghai, China), the membranes were incubated with rabbit anti-human mTOR primary antibody (1:400; sc-8319) overnight at 4°C, then with secondary horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5,000; sc-2004; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. The immunoreactive bands were detected using an enhanced chemiluminescence kit (PI-32209; Pierce Biotechnology, Inc., Rockford, IL, USA). β-actin was used as an internal control. The protein concentrations were assessed using a bicinchoninic acid protein assay kit (Beijing Comwin Biotech Co., Ltd.). BCA solution (200 µl) was added to each well and allowed to stand at 37°C for 30 min. The OD value of each well was read at a wavelength of 562 nm using a microplate reader (Model 680, Bio-Rad Laboratories Inc.). A standard curve was drawn and the protein concentration was calculated.

The CCK-8 kit was used to monitor cell proliferation. Briefly, SMMC-7721 cells were plated at a density of 1×104 cells/well in 96-well plates. Three groups were established, including an experimental group (transfection + hyperthermia), a control group (hyperthermia) and a blank control group. In the experimental group, the cells were subjected to heat shock in an incubator at 42°C for 1 h post-transfection. Cells were cultured at 42°C for 1 h without transfection in the hyperthermia group. The blank control group cells were incubated at 37°C without any treatment. The absorbance of the cells was determined at a wavelength of 450 nm according to the manufacturer's protocol. Experiments were conducted in triplicate and the average results were calculated.

SMMC-7721 cells were seeded at an initial density of 5×104 cells/well on 24-well plates and cultured to 80% confluence. Following treatment, the cells were wounded by manual scraping with a sterile 10-µl pipette tip. The culture medium was then replaced with fresh, FBS-free DMEM. Wound closure was monitored at various time points by observation under an inverted microscope, and images were captured at regular time intervals (TE2000-U; Nikon Corporation). Wound distances were measured at each time point and expressed as the average percentage of wound closure compared with that at 0 h.

SMMC-7721 cells were divided into three groups as described above. Cells were collected following hyperthermia treatment and reseeded in 6-well plates (1×103 cells/well), each group contained three wells. Following incubation at 37°C for 14 days, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min and stained with 2 ml Giemsa reagent for 30 min (Hematology Laboratory, the Third Affiliated Hospital of Sun Yat-Sen University). The number of colonies containing 50 cells was counted manually under low magnification. Clone formation efficiency was calculated using the following formula: Clone formation efficiency = (number of colonies/number of cells inoculated) × 100%.

SMMC-7721 cells at an initial density of 5×104 cells/well were incubated in 24-well plates and treated as mentioned previously. Cells with different treatments were collected separately and resuspended with PBS. Subsequently, cells (5×10⁵) were centrifuged at 500 × g for 5 min and the supernatant solutions were discarded. An Annexin V-PE/7-AAD Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA) was used according to the manufacturer's protocol. After adding 50 µl binding buffer and 5 µl 7-AAD, cells were incubated at room temperature for 15 min in the dark. After that, cells were resuspended with 450 µl binding buffer and 1 µl Annexin V-PE was added. The fluorescence of Annexin V-PE and 7-AAD was measured by flow cytometry (FACSCalibur; BD Biosciences). For cell cycle analysis, cells were collected, washed twice with PBS and incubated with ice-cold 70% ethanol overnight. Subsequently, the fixed cells were resuspended in PBS and stained with 50 µg/ml propidium iodide (Sigma-Aldrich) at room temperature in the dark for 30 min. Flow cytometry analysis was performed using the FACSCalibur cytometer.

Statistical analysis was performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). All results were presented as means ± standard deviation. One-way analysis of variance was used to analyze the statistical differences between multiple groups. P<0.05 was considered to indicate a statistically significant result.

It has been reported that cancer cells are susceptible to hyperthermia (15). To detect the effects of hyperthermia on SMMC-7721 cells, the cells were exposed to heat treatment at various temperatures. Following hyperthermia, folding of the cell membrane was observed and numerous vacuoles formed in the cytoplasm. As the temperature increased, the number of necrotic cells also increased (Fig. 1A). Hyperthermia significantly inhibited the growth of SMMC-7721 cells compared with cells at 37°C (P<0.01). The inhibitory rate of cells at 44°C was increased by 40.65%. Hyperthermia reduced cell proliferation in a temperature-dependent manner (Fig. 1B). SMMC-7721 cell viability was significantly decreased at 42°C compared with cells at 37°C after 96 h treatment (Fig. 1C).

Following antisense plasmid pEGFP-C1-mTOR transfection into the SMMC-7721 cells, the expression of green fluorescent protein was observed using inverted fluorescent microscopy (Fig. 2A). The results from the RT-PCR analysis demonstrated that the expression of mTOR mRNA was significantly reduced post-transfection (P<0.01). The relative values of the two PCR assays were 0.74±0.04 and 0.23±0.01 (Fig. 2B). The protein expression levels of mTOR were detected using western blotting and demonstrated similar results. The relative values of mTOR protein expression were 0.65±0.03 and 0.31±0.02, respectively. These results indicated that mTOR protein expression was also significantly decreased post-transfection (P<0.05; Fig. 2C).

CCK-8 analysis demonstrated a significant reduction in cell viability of the experimental group at 24 and 48 h compared with the other groups (Fig. 3). The wound-healing assay demonstrated that numerous cells migrated into the scratch wound area in the control and blank control groups, whereas there were fewer cells in the experimental group (Fig. 4). Furthermore, a larger number of necrotic cells was also observed in the experimental group. These results indicated that cell migration was reduced following treatment. The colony forming assay determined that the clone formation efficiencies for the control and blank control groups were 9.05±1.97 and 14.5±2.45%, respectively. The number of cell colonies was markedly decreased in the experimental group, and the clone formation efficiency was 1.98±0.61% (Fig. 5).

The present study examined whether the change in cell proliferation was associated with the apoptosis induced by heat. As shown in Fig. 6A, the rate of apoptosis for the control and blank control groups was 10.14±2.66 and 28.93±2.51%, respectively. The apoptotic rate in the experimental group was significantly lower compared with that in the other two groups (52.27±3.72%; P<0.01). This result suggests that mTOR expression protects SMMC-7721 cells from heat-induced apoptosis, and thus increases cell survival following exposure to heat. To determine whether the cell cycle was among the mechanisms underlying the change in cell proliferation, flow cytometric analysis was performed. Compared with the other two groups, the number of cells in the G₁ phase was decreased in the experimental group. However the proportion of cells in S phase was increased (Fig. 6B). These results suggest that the transfected cells were arrested in S phase following hyperthermia.