Human normal hepatocyte (L02) and HCC cell lines (Huh7, HepG2, Hep3B and SMMC-7721) were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in a petri-dish (Corning, Shanghai, China) containing Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.; Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.), in an atmosphere containing 5% CO₂ at 37°C.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The ultramicro spectrophotometer (Thermo Fisher Scientific, Inc.) was used to detect the purification and quantification of RNA. Complementary DNA (cDNA) was synthesized using PrimeScript™ RT reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China). DNase (Takara Biotechnology Co., Ltd.) was used prior to the reverse transcription reaction. The conditions for reverse transcription reaction were as follows: 37°C for 15 min and then 85°C for 5 sec. RT-qPCR was performed using the Bio-Rad iCycler iQ Real-Time PCR system (Bio-Rad, Hercules, CA, USA) with the following primers: HSP70 forward, 5′-ACCTACTCCGACAACCAA-3′ and reverse, 5′-AGATGACCTCTTGACACTTG-3′; GAPDH forward, 5′-CATGGCAAATTCCATGGCA-3′ and reverse, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′. The PCR mixture was prepared using SYBR^® Master Mix (Takara Biotechnology Co., Ltd.) in accordance with the protocol of the manufacturer. The conditions for PCR reaction were as follows, 95°C for 30 sec for one cycle, and then 95°C for 5 sec, 60°C for 30 sec for 40 cycles. To ensure that only specific bands were produced, the melting curve was analyzed at the end of each PCR experiment. Expression levels of each mRNA were determined using the 2^−ΔΔCq method using GAPDH as an endogenous control (14).

For western blotting, cells were lysed for 30 min on ice with radioimmunoprecipitation assay buffer containing phosphatase and protease inhibitors (Beyotime, Haimen, Jiangsu, China). The cell lysate was then centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was carefully collected following centrifugation. The protein concentrations were determined using a bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). The cell lysates (50 µg protein/lane) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (HyClone; GE Healthcare Life Sciences). The membranes were blocked with 5% (v/v) skim milk at room temperature for 1 h, and then probed with the primary antibodies at 4°C overnight. Following washing with TBS/Tween-20 three times, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (cat. no. 7076; Cell Signalling Technology, Danvers, MA, USA; dilution, 1:1,000;) at room temperature for 1 h. A primary antibody against cyclin-dependent kinase inhibitor 1B (p27Kip1) was purchased from BD Biosciences (cat. no. 610241; San Jose, CA, USA; dilution, 1:2,000). A primary antibody against Cyclin D1 was purchased from Santa Cruz Biotechnology Inc. (cat. no. sc-4074; Dallas, TX, USA; dilution, 1:1,000). Relative optical density of all bands was analyzed by GelPro Analyzer (V4.0; Media Cybernetics, Rockville, MD, USA).

Cell apoptosis was evaluated with the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (cat. no. 556547; BD Biosciences) according to the manufacturer's protocol. Pre-treated Hep3B or SMMC-7721 cells were stained with FITC-Annexin V and PI, and then evaluated for apoptosis using flow cytometry (FACSCaliburTM; BD Biosciences), according to the manufacturer's protocol. Briefly, cells were harvested following the incubation period, washed twice in cold PBS, and centrifuged at 300 × g for 5 min at 4°C. A total of ~1×106 cells were re-suspended in 500 µl 1X Annexin-binding buffer and transferred to a sterile flow cytometry glass tube. A total of 5 µl FITC-Annexin V and 10 µl PI were added to each tube and incubated at room temperature for 5 min in the dark. Subsequent to incubation, samples were analyzed using the standard program of flow cytometry and analyzed using FlowJo V7.6.1 software (Tree Star, Inc., Ashland, OR, USA).

The viability of SMMC-7721 and Hep3B cells post-transfection was determined using a Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells were seeded in 96-well culture plates at a density of 4×103 cells per well and transfected with the desired small interfering (si)RNA (GenePharma, Shanghai, China). Cellular transfection was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the protocol. Subsequent to culturing for 1–5 days at 37°C with 5% CO₂, the supernatant was removed and cell growth was determined using a CCK-8 kit, according to the manufacturer's protocol. Absorbance was measured at 450 nm using a microplate reader.

Cells (1×106 cells/well) were seeded on 6 well plates (Corning Life Sciences, Shanghai, China) and cultured with DMEM medium at 37°C with 5% CO₂ for 24 h, prior to being transfected with 100 nM HSP70-siRNA or control siRNA (siCON) for 48 h. For the flow cytometry, cells were trypsinized, pelleted via centrifugation at 1,000 × g for 5 min at 4°C and resuspended in 300 µl 0.1% Triton X-100 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)/PBS. Cells were fixed with the cold 70% ethanol (Sangon Biotech Co., Ltd., Shanghai, China) at 4°C for 2 h and centrifuged at 1,000 × g for 5 min at 4°C. Subsequent to washing twice in cold PBS and centrifugation at 1,000 × g for 5 min at 4°C, cells were treated with RNase Type I-A (Sigma-Aldrich; Merck KGaA) at 37°C for 15 min and stained with PI (1 mg/ml) at room temperature for 10 min in the dark. Cellular DNA content was determined using a FACSCalibur™ (BD Biosciences). Cell cycle phase distributions were analyzed by ModFit LT™ (v3.0; BD Biosciences) cell cycle analysis software.

For the migration assay, 1×104 Hep3B or SMMC-7721 cells were plated into 24-well Boyden chambers (Corning Incorporated, Corning, NY, USA) with an 8-µm pore polycarbonate membrane. For the invasion assay, 1×104 Hep3B or SMMC-7721 cells were plated on chambers pre-coated with 20 µg Matrigel. In these two assays, the cells were plated in DMEM medium without serum in the upper chamber, and medium containing 10% fetal bovine serum in the lower chamber served as a chemoattractant. After 24 h, cotton swabs removed the cells that did not migrate or invade through the pores. The inserts were fixed with 4% paraformaldehyde (Sangon, Shanghai, China) (30 min at room temperature), stained with 0.1% crystal violet (Beyotime, Haimen, Jiangsu, China) (30 min at room temperature) and washed with PBS (room temperature). Five random fields for each insert were counted under an inverted microscope at ×100 magnification (Olympus Corporation, Tokyo, Japan).

All statistical analyses were performed using SPSS v18.0 (SPSS Inc., Chicago, IL, USA). The results are presented as the means ± standard deviation, and statistical analysis was performed using the unpaired Student's t-test for two groups and one-way analysis of variance for more than two groups, followed by Dunnett's Test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference. At least three replicates were performed for each experiment.

To demonstrate the expression of HSP70 in cell lines, normal hepatocyte L02 and four HCC cell lines (Huh7, HepG2, SMMC-7721 and Hep3B) were used. In contrast to normal L02 hepatocytes, the four HCC cell lines exhibited greater expression of HSP70 (Fig. 1A), suggesting that HSP70 may be involved in hepatocarcinogenesis. Subsequently, to elucidate the role of HSP70 in hepatocarcinogenesis, the effect of HSP70 knockdown on HCC cell growth following transfecting HCC cells with HSP70-siRNA or siCON was examined. In the present study, two HCC cell lines (SMMC-7721 and Hep3B) were selected as the cell models, as they expressed higher levels of HSP70 when compared with the other two cell lines. The results of the western blotting and qPCR demonstrated that HSP70-siRNA may effectively suppress the protein and mRNA levels of HSP70 in the two examined cell lines (Fig. 1B and C).

The biological role of HSP70 in the growth of HCC cells was subsequently investigated. The proliferative capacity of SMMC-7721 (Fig. 2A, left) and Hep3B (Fig. 2A, right) cells following HSP70 knockdown decreased markedly when compared with that of corresponding siCON-transfected control cells, particularly from day 3–5, as detected using a CCK-8. Furthermore, the cloning efficiency in the siCON group was significantly greater compared with that in HSP70-siRNA groups of SMMC-7721 (Fig. 2B, left; P<0.05) and Hep3B (Fig. 2B, left; P<0.05). Therefore, transfection with HSP70-siRNA markedly reduced the proliferative ability of the two HCC cell lines, which is concordant with the aforementioned CCK-8 assay results.

To further understand the mechanism underlying HSP70-knockdown-induced cell growth inhibition, flow cytometry was performed to examine the cell cycle of SMMC-7721 and Hep3B cells transfected with HSP70-siRNA or siCON for 48 h. The proportion of the two cell lines treated with HSP70-siRNA in the G₀/G1 stage evidently increased, while those in the S and G2 stages markedly decreased with respect to the siCON and untreated groups. In addition, the cell cycle distribution of HCC cells in the siCON and the untreated group was similar (Fig. 3A and B; P<0.05), and no significant difference was observed. These results suggested that the growth inhibition induced by HSP70 downregulation is mediated by cell cycle arrest at the G1/S phase in HCC cells. Additionally, cyclin D1 and p27Kip1, two cell cycle-associated molecules, were used to evaluate the differences in the cell cycle distribution of the two cells. Cyclin D1 expression was notably decreased, whereas p27Kip1 expression was markedly increased, in the HSP70-siRNA group, as compared with in the siCON and untreated groups (Fig. 3C).

As recurrence and metastasis are two main factors contributing to the poor prognosis of HCC, the migratory and invasive ability of SMMC-7721 and Hep3B cells was examined. The HSP70-siRNA of SMMC-7721 (Fig. 4A) and Hep3B (Fig. 4B) cells significantly inhibited migration compared with control groups after 48 h. In the Transwell migration and invasion assay (Fig. 4C and D), HSP70 knockdown decreased cell migration in the two tested cell lines, compared with in the siCON and control groups. Furthermore, there a higher number of invaded cells in the siCON and control groups, as compared with those in the HSP70-siRNA group. Consistently, knockdown of HSP70 inhibited cell migration and invasion. Thus, the data indicate that an overexpression of HSP70 promotes migration and invasive capabilities in vitro, and a reduction of HSP70 correspondingly attenuates HCC cell aggression.