HepG2 cells were purchased from American Type Culture Collection (cat. no. HB 8065). HL-7702 cells were purchased from cell bank of typical culture preservation Committee of Chinese Academy of Sciences. The cells were cultured at 37°C in an atmosphere containing 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; Jiangsu Kaiji Biotechnology Co., Ltd.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) for different durations (2, 6, 12, 24 and 48 h). The cell line was authenticated by short tandem repeat analysis.

In this experiment, the FX-4000T™ Tension system (Flexcell International Corporation) purchased by the Biomechanics Laboratory of Shanghai Jiao Tong University was used. The tensile strain principle of the device was to use a vacuum pump to draw air, make space under an elastic film in the vacuum environment and produce downward tension strain. When the vacuum pump was relaxed, the elastic membrane retracted under its own elastic force and circulated, thus producing periodic tensile strain (14).

Numerous routine treatment methods were tested in the laboratory, such as soaking in hydrochloric acid (plate) for 24 h, followed by soaking in water for 24 h, then drying, gelatin coating or cleaning. Moreover, drying after soaking in 75% alcohol for 24 h, then gelatin coating was performed. However, when using these methods cells did not adherent to the wells and grew poorly. Later, the plate was washed and dried. Then, the gelatin was changed into rat tail collagen for coating, it was found that the cells could adhere to the wall and grow efficiently. The BioFlex amino-culture plates (Flexcell International Corporation) were coated with 2 mg/ml rat tail collagen solution (Roche Diagnostics). Then, 20 µl of collagen solution was added to each well and smeared evenly with an aseptic cell scraper. Subsequently, the plates were blow-dried on a clean bench and irradiated with ultraviolet light for 1 h.

The cells were seeded on BioFlex amino-culture plates at a density of 1×10⁵ cells/well. After 24 h, the culture medium was replaced with serum-free medium for another 24 h to synchronize the cells in 37°C incubator. The cells in the serum-free medium were then exposed to cyclic stretch provided by the FX-4000T Tension system.

In the periodic strain-loaded cells, three main parameters were used; these were tensile strain amplitude, tension strain frequency and time. By fixing two of the parameters, the other parameter was changed to observe the effect of tension strain on HepG2 cells (except these three parameters, all the other experimental conditions were the same). After synchronizing HepG2 cells implanted on BioFlex amino-culture plates for 24 h, three groups of experiments were set up: i) Tension strain time (tensile strain frequency fixed to 1 Hz; amplitude to 15%; and tension strain time set to 2, 6, 12, 24 or 48 h, a total of five experimental groups); ii) tensional strain frequency (tensile strain time fixed to 24 h; amplitude to 15%; and tensile strain frequency set to 0.5 and 1 Hz, a total of two experimental groups); and iii) tensile strain amplitude (tensile strain time fixed to 24 h; frequency to 1 Hz; and tensile strain amplitude set to 5 and 15%, a total of two experimental groups). All the control groups were nontension loaded groups. After tension loading, the cells were digested with trypsin (containing EDTA; Jiangsu Kaiji Biotechnology Co., Ltd.), resuspended with 0.5 ml 1X PBS containing 50 µg/ml propidium iodide (PI) and 200 µg/ml ribonuclease (Roche Diagnostics) and incubated at 37°C for 30 min. PI is an insertion nucleic acid fluorescent dye; its binding amount is proportional to the content of DNA in the cell. Using flow cytometry and FlowJo V10 software (FlowJo LLC) DNA distribution status at various stages of the cell cycle can be obtained, and the percentage of each cell cycle can be calculated. After propidium iodide staining, if the fluorescence intensity of G0/G1 phase cells is 1, the theoretical value of the fluorescence intensity of G2/M phase cells that contain double genomic DNA is 2, and the fluorescence intensity of S phase cells undergoing DNA replication is 1–2 (15). Then, flow cytometry was used to detect the cell cycle with a standard program. In the analysis, the proliferation index (PI) = (S + G2/M) / (G0/G1 + S + G2/M) was used to evaluate the effect of different tensile strain loading conditions on cell proliferation, and the ideal tension strain conditions for HepG2 cells were determined. The experiment was repeated three times.

The HepG2 cells and HL-7702 cells seeded on the BioFlex amino-culture plates were synchronized for 24 h and then connected to the Flexcell FX-4000T Tension system. The experimental group was treated for 24 h with a tensile strain amplitude of 15% and a frequency of 1 Hz, while the control group was the non-tension loaded group. BrdU (2 µl/well; Sigma-Aldrich; Merck KGaA) was added to the tension strain and control groups simultaneously at 8 h before the end of the tension strain treatment in the 37°C incubator. At the end of the strain, the cells were collected by trypsin digestion, and the cell concentration was adjusted to 1×10⁵/ml in DMEM (containing 10% FBS). The cells were cultured on the 96-cell plate (100 µl of cell suspensions/well) at 37°C in an atmosphere containing 5% CO₂ for 4 h until they adhered. Finally, the medium was dried and put in a 4°C refrigerator overnight. Furthermore, FixDenat solution (200 µl/well; Sigma-Aldrich; Merck KGaA) was added and incubated for 30 min at room temperature. The anti-BrdU-Peroxidase storage solution was diluted 100 times with antibody diluent, added to the cells (100 µl/well) and incubated at room temperature for 90 min. The supernatant was removed, and PBS was used to wash the cells three times. Next, tetramethylbenzidine substrate (100 µl/well; Sigma-Aldrich; Merck KGaA) was added at room temperature for 5–30 min to complete color development. Subsequently, sulfuric acid (1 M) was added to terminate the reaction immediately (25 µl/well). The 96-cell plate was vibrated for 60 sec and the absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.), with a reference wavelength of 630 nm.

The HepG2 cells seeded on the BioFlex amino-culture plates were synchronized for 24 h and then connected to the Flexcell FX-4000T Tension system. The experimental group was treated for 24 h with a tensile strain amplitude of 15% and a frequency of 1 Hz, while the control group was the nontension loaded group. Then, 0.5 h before the end of the tension strain treatment, CCK-8 reagent (Sangon Biotech, Co., Ltd.) was added to the tension strain and control groups at the same time (200 µl/well), and the assay was operated according to the manufacturer's protocols. After the tension strain treatment, the cell culture medium in each well was transferred into a 96-well plate. Subsequently, the 96-cell plate was put in the microplate reader (Bio-Rad Laboratories, Inc.). After vibration for 60 sec, the absorbance at 450 nm was measured, with a reference wavelength of 630 nm.

Total RNA was extracted by TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, purified using miRNeasy Mini kit (Qiagen GmbH) and quantified using the NanoDrop™ 2000 (Thermo Fisher Scientific, Inc.), and the RNA integrity was assessed using an Agilent 2100 Bioanalyzer instrument (Agilent Technologies, Inc.). The sample labeling, microarray hybridization and washing were performed based on the manufacturer's standard protocols. Differentially expressed miRNAs were then identified based on fold-change and P-value, as calculated using the t-test. The threshold set for up- and downregulated genes was a fold-change ≥2.0 and P<0.05.

RT-qPCR was performed to validate the microarray data. The extracted total RNAs were reverse transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Thermo Fisher Scientific, Inc.) with a special stem-loop primer (RT primer). The thermocycling conditions were as follows: 42°C for 1 h, followed by 70°C for 5 min. Then, RT-qPCR was performed with a SYBR Green PCR kit (Takara Bio, Inc.) on an ABI PRISM 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The thermocycling conditions were as follows: Initial denaturation at 95°C for 15 min, followed by 40 cycles at 62°C for 40 sec, 95°C for 1 min and 55°C for 1 min. The sequences of RT and PCR primers are listed in Table I. The expression levels of miRNAs were normalized to U6 expression. miRNA expression levels were calculated using the 2^−ΔΔCq method (16).

Target genes of differentially expressed miRNAs were determined as the intersection of the predictions of the three databases: TargetScan V7.2 (http://www.targetscan.org/), microRA.org (http://www.microrna.org; November 2010 version) and PITA (https://genie.weizmann.ac.il/) (17). All data were obtained from the databases in January 2019.

GO analysis (http://geneontology.org/docs/go-citation-policy/) and pathway analysis were performed on predicted mRNA targets of miRNAs using Database for Annotation, Visualization, and Integrated Discovery (18) and KEGG database (19,20), respectively. Specific biological process categories and pathways were enriched. The threshold of significance was defined by P<0.05 and Benjamini-Hochberg (21) false discovery rate (FDR-bh) <0.25.

Statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software, Inc.). Each experiment was performed at least three times, and all values were expressed as the mean ± standard deviation. Data from ≥3 were analyzed using one-way ANOVA followed by Fisher's Least Significant Difference or Dunnett's post hoc comparison test as applicable. P<0.05 was considered to indicate a statistically significant difference.

Due to the lack of related reports on HepG2 cells cultured on BioFlex plates, several methods were tried in this experiment. Finally, type I rat tail collagen was selected. The results showed that type I rat tail collagen coating led to increased cell adherence and growth of HepG2 cells in the BioFlex plates (Fig. 1).

i) Tension strain time: The PI increased significantly in 24 and 48 h groups compared with the control group without tension (P<0.0001; Fig. 2A and B). ii) Tensional strain frequency: The PI increased significantly in the 1 Hz group compared with the control group without tension (P<0.001; Fig. 2C and D). iii) Tensile strain amplitude: The PI increased significantly in the 15% group compared with the control group without tension (P<0.0001; Fig. 2E and F).

Based on the experimental results of the aforementioned three groups of tensile strain loading conditions, a tensile strain amplitude of 15%, frequency of 1 Hz and time of 24 h were selected as the ideal conditions for the following experiments.

The results of the BrdU assay showed that the tensile strain amplitude of 15% at 1 Hz for 24 h significantly promoted DNA synthesis in cells compared with the control group (P<0.001; Fig. 3A). Additionally, the CCK-8 assay also demonstrated that these conditions significantly promoted DNA synthesis in the cells (P<0.0001; Fig. 3B). Thus, this indicated that tensile strain could lead to increased liver cancer cell proliferation.

To investigate the effects of tensile strain on the miRNA profile of HepG2 cells, tensile strain was applied to the activated HepG2 cells, and RNA was extracted and subjected to microarray analysis. As shown in Table II, seven significantly differentially expressed miRNAs showed a fold-change threshold of ≥2.0 and P<0.05. Of these seven miRNAs in the tensile strain-treated group, five were upregulated (hsa-miR-296-5p, hsa-miR-6752-5p, hsa-miR-6794-5p, hsa-miR-6889-5p and hsa-miR-7845-5p) and two were downregulated (hsa-miR-4428 and hsa-miR-503-5p) compared with the control group.

RT-qPCR was performed to validate the microarray results of the five upregulated and two downregulated miRNAs. As shown in Fig. 4, the changes in the seven miRNAs detected by RT-qPCR were consistent with those of the miRNA microarray.

Potential target genes for the aforementioned seven differentially expressed miRNAs were searched using three bioinformatics databases, miRNAorg, PITA and TargetScan, which predicted 2,306, 1,277 and 16,205 target genes, respectively. The false results were removed by taking the intersection of the results from the three databases for 224 target genes (Fig. 5). The 16,205 predictive target genes refer to the sum of all seven predictive target genes that differentially express miRNAs. The components are shown in the Table III.

GO and pathway analyses were used to understand the biological characteristics of the aforementioned target genes. Using a threshold of FDR-bh <0.25, 132 GO terms were considered to be significant. Additionally, 6, 25 and 101 GO terms belonged to the ‘cellular component’ category (Fig. 6A), ‘molecular function’ category (Fig. 6B) and ‘biological process’ category (Fig. 6C), respectively. The x-axis of Fig. 6 shows the P-value corrected by FDR-bh algorithm, which is converted by -log10. The longer the column is, the higher the enrichment degree is. The target genes were concentrated in ‘cyclin-dependent protein kinase holoenzyme complex’, ‘neuromuscular junction’, ‘microtubule cytoskeleton’, ‘protein kinase binding’, ‘protein binding’, ‘positive regulation of transcription from RNA polymerase II promoter’, ‘positive regulation of transcription, DNA-templated’, ‘negative regulation of cell migration’, ‘positive regulation of skeletal muscle tissue development’ and ‘patterning of blood vessel’ categories.

Using KEGG pathway analysis, 52 significant KEGG pathways were identified at a cutoff FDR-bh <0.25. These pathways included diseases, such as ‘breast cancer’, ‘melanoma’, ‘prostate cancer’, ‘endocrine resistance’ and ‘bladder cancer’, and signaling pathways, such as ‘mitogen-activated protein kinase (MAPK) signaling pathway’, ‘PI3K-Akt signaling pathway’, ‘forkhead box O signaling pathway’ and ‘Ras signaling pathway’ (Fig. 6D).

Using BrdU experiments, it was demonstrated that the proliferative ability of HepG2 cells after tension intervention was significantly improved (P<0.05), while the proliferative ability of normal hepatocyte HL-7702 did not change significantly (Fig. 7).

Flexcell-5000T was also used to performed traction experiments on Huh-7 cells and MHCC97H cells, but the two cells could not adhere to the membrane on the traction plate, and thus HepG2 cells were selected for subsequent experimentation.