HL-7702, MIHA, Hep3B, Huh-7, MHCC-97H, MHCC-97L, MHCC-LM6, MHCC-LM3, YY-8103, SK-hep-1 cell lines were all obtained from the Type Culture Collection of the Chinese Academy of Sciences. The HL-7702 cell line is a normal liver cell line, and was used as a control group. HL-7702 and MIHA cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.); Hep3B, Huh-7, MHCC-97H, MHCC-97L, MHCC-LM6, MHCC-LM3, YY-8103, SK-hep-1 cell lines were cultured in DMEM (Thermo Fisher Scientific, Inc.). Medium was supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences), and cells were cultured at 37°C with 5% CO₂. Cells were subcultured every 2–3 days. The SIRT6 overexpression plasmid, the negative control (NC) plasmid (empty pcDNA3.1 vector), the plasmid containing small interfering RNA against SIRT6 (siSIRT6) and the plasmid containing NC-siRNA were synthesized by Shanghai GenePharma Co., Ltd. The sequences of oligonucleotides were as follow: siSIRT6, 5′-TCATGACCCGGCTCATGAA-3′; NC-siRNA, 5′-TCACCCATCGGTACGTGAA-3′. Huh-7 cell line was divided into 3 groups: Control (blank); NC or siNC (cells transfected with the NC plasmid or NC-siRNA plasmid); and SIRT6 overexpression or siSIRT6 groups (cells transfected with the SIRT6 overexpression or siSIRT6 plasmid). To further explore whether SIRT6 affects the proliferation and apoptosis of HCC cells by regulating the ERK1/2 pathway, the cells were treated with 10 µM U0126 at 37°C for 24 h, and U0126 treatment and transfection occurred simultaneously. Huh-7 cells were also separated into control (blank), NC (cells transfected with the NC plasmid), SIRT6 (cells transfected with the SIRT6 overexpression plasmid), U0126 (cells treated with 10 µM U0126), SIRT6 + U0126 (cells transfected with the SIRT6 overexpression plasmid and treated with 10 µM U0126) groups for the measurement of cell proliferation using ERK1/2 inhibitor U0126. Transfection was conducted using Lipofectamine™ 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Following transfection for 48 h, cells were used to subsequent experiments.

Cell proliferation was assayed using a Cell Counting Kit-8 (Sigma-Aldrich; Merck KGaA) at 0, 24 and 48 h following transfection. Procedures were performed according to the manufacturer's protocols. Cells (3×10³ cells/well) were then incubated in a 96-well plate for 1 h at 37°C, and the optical density (OD) was measured at 450 nm using a microplate reader (Multiskan™; Thermo Fisher Scientific, Inc.).

Following transfection for 48 h, Huh-7 cells were washed with PBS and dissociated with 0.25% trypsin for 2 min at 37°C. Cells (1–5×10⁵) were centrifuged at 800 × g for 5 min and collected. Using an Annexin V-FITC Apoptosis Staining/Detection kit (Abcam), 195 µl Annexin V binding solution, 6 µl Annexin V and 4 µl propidium iodide were added to cells, and then mixed gently by pipetting. Cells were incubated at room temperature for 20 min in the dark, and immediately analyzed using a flow cytometer (BD FACSCanto II; BD Biosciences) and FACSDiva software version 6.1.2 (BD Biosciences) to detect the apoptosis rate. Flow cytometry demonstrated that the advanced apoptotic cells were in the upper right quadrant, and the early apoptotic cells were in the lower right quadrant. The apoptotic rate was the sum of the early and advanced apoptotic rates.

Transfected cells were collected and inoculated into a 100-mm dish at a concentration of 200 cells/dish following transfection for 48 h. G418 (700 µg/ml; Abcam) was added to the medium and mixed to detect positive cell clones for 3 weeks until visible cell clones emerged. Fresh medium was replaced every 3 days. The supernatant was then discarded, and the cells were gently washed with PBS twice. Cells were fixed with methanol for 15 min at room temperature, and stained with crystal violet for 10–30 min at room temperature. Cells were then washed slowly with running water and dried. Each cell clone on the dishes was counted and photographed under a light microscope (magnification, ×40).

Total RNA was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) and 1 µg of RNA of each sample was transcribed into cDNA using an iScript™ cDNA Synthesis kit (Bio-Rad Laboratories, Inc.). The reverse transcription reaction was performed at 42°C for 15 min, followed by reverse transcriptase inactivation at 85°C for 15 sec. A Fast Start Universal SYBR-Green Master kit (Roche Diagnostics) was used to perform qPCR. The primers used for the reaction are presented in Table I. The reaction system was the following: 2X SYBR-Green Master Mix (12.5 µl); cDNA template (2 µl); forward primer (10 µM; 1 µl); reverse primer (10 µM; 1 µl); and ddH₂O (8.5 µl). qPCR was conducted as follows: 95°C for 10 min, then 40 cycles of 95°C for 15 sec, 60°C for 1 min and 72°C for 3 min. qPCR was conducted in a CFX96 Touch™ system (cat. no. 6093; Bio-Rad Laboratories, Inc.). The 2^−ΔΔCq method (15) was used to calculate the relative mRNA expression in samples; GAPDH was used as an internal reference.

Cells were collected and washed with PBS, and RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) was added to lyse the cells according to the manufacturer's protocols. Cells were then centrifuged at 4°C and 16,000 × g for 15 min to remove the cell debris and supernatant, and total protein was collected. The concentration of total protein was determined using a Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Standard protein was diluted to 1, 0.5, 0.25, 0.125 and 0.0625 g/ml respectively, then 2 µl samples and standard proteins were added to a 96-well plate. BCA reagent was subsequently added, and plates were incubated at 37°C for 30 min. The OD at 562 nm was detected using a microplate reader. A standard curve was drawn, and the concentration of the total protein was then calculated.

Total protein (30 µg) from each sample was denatured at 95°C for 10 min, and proteins were separated via 10% SDS-PAGE at 100 V for 2 h. Proteins were transferred onto PVDF membranes using a wet transfer electrophoresis tank (Bio-Rad Laboratories, Inc.) at 90 V for 2 h, and then blocked in 5% non-fat milk for 1 h at room temperature. Membranes were then incubated overnight at 4°C with the following primary antibodies: Anti-SIRT6 (1:2,000; cat. no. ab191385; Abcam); anti-cleaved-caspase-3 (Asp175; 1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.); anti-Bcl-2 (1:2,000; cat. no. ab32124; Abcam); anti-Bax (1:2,000; cat. no. ab32503; Abcam); anti-GAPDH (1:2,000; cat. no. ab181602; Abcam); anti-ERK1/2 (1:2,000; cat. no. ab17942; Abcam); and anti-phosphorylated (p)-ERK1 (T202) + ERK2 (T185; 1:2,000; cat. no. ab201015; Abcam). Membranes were washed three times with PBS-0.05% Tween 20 (PBST; Beijing Solarbio Science & Technology Co., Ltd.) for 5 min, and the horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. ab6721; Abcam) was added. Membranes were then incubated for 1 h at RT, and bands were visualized using Pierce™ ECL Plus western blotting substrate (Thermo Fisher Scientific, Inc.) following three washes in PBST. The densitometry was performed using the Bio-Rad ChemiDoc system with Image Lab software version 6.0 (Bio-Rad Laboratories, Inc.).

Data were analyzed using GraphPad Prism 7.0 software (GraphPad Software, Inc.). All data were presented as the mean ± standard deviation. One-way ANOVA followed by Tukey's post hoc test were used to compare between groups. P<0.05 was considered to indicate statistical significance.

To investigate the expression of SIRT6 in HCC cell lines, MIHA, HL7702, Hep3B, Huh-7, MHCC-97H, MHCC-97L, MHCC-LM6, MHCC-LM3, YY-8103 and SK-hep-1 cell lines were purchased, and the mRNA expression of SIRT6 was measured via RT-qPCR analysis. As presented in Fig. 1, the expression of SIRT6 mRNA in Hep3B, Huh-7, MHCC-97H, MHCC-97L, MHCC-LM6, MHCC-LM3, YY-8103 and SK-hep-1 cells was significantly upregulated compared with HL-7702 cells, normal human liver cells set as the control (P<0.01). The results indicated that the expression of SIRT6 was elevated in HCC.

To investigate the effects of upregulated expression of SIRT6 on the proliferation and apoptosis of HCC cells, the proliferation, cloning efficiency and apoptosis of Huh-7 cells was evaluated following overexpression of SIRT6. Overexpression of SIRT6 mRNA and protein was demonstrated in Huh-7 cells (P<0.01; Fig. 2A and B), and the proliferation of transfected Huh-7 cells was significantly increased compared with the control or NC groups at 24 and 48 h following transfection (P<0.05; Fig. 2C). Cloning efficiency was increased in the SIRT6 overexpression group compared with the control or NC groups (P<0.01; Fig. 2E); however, the apoptosis rate was not significantly affected by SIRT6 overexpression (Fig. 2D). The results suggested that overexpression of SIRT6 may promote the proliferation of HCC; however, its effects on apoptosis require further investigation.

To further determine the role of SIRT6 in HCC, the effects of silencing SIRT6 on the proliferation and apoptosis of Huh-7 cells was investigated. It was revealed that SIRT6 expression was suppressed in Huh-7 cells following transfection with siSIRT6 (P<0.01; Fig. 3A and B). Additionally, the proliferation of cells in the siSIRT6 group was significantly decreased compared with the control and si-NC groups at 24 and 48 h (P<0.05; Fig. 3C). Furthermore, the cloning efficiency of siSIRT6-transfected cells was significantly reduced compared with the control and si-NC groups as well (P<0.01; Fig. 3E). The apoptosis rate was also significantly increased in siSIRT6 the group compared with the controls (P<0.01; Fig. 3D). Collectively, the results indicated that the downregulation of SIRT6 decreased the proliferation and increased the apoptosis of HCC cells, opposing effects to the results observed following the overexpression of SIRT6.

To evaluate the activity of the intrinsic apoptosis pathway, the mRNA expression of Bcl-2 and Bax was determined via RT-qPCR analysis, and protein levels of Bcl-2, Bax and cleaved-caspase-3 were measured via western blotting. It was revealed that the expression of Bcl-2 in the SIRT6 overexpression group was significantly upregulated compared with the control or NC groups, and the expression of Bax and cleaved-caspase-3 was significantly downregulated (P<0.05; Fig. 4A, C and E). Opposing results were observed following siSIRT6-mediated knockdown of SIRT6 (P<0.01; Fig. 4B, D and F). The results indicated that overexpression of SIRT6 downregulated the intrinsic apoptosis pathway in HCC cells, whereas silencing SIRT6 induced opposing effects.

To investigate the mechanisms underlying the effects of SIRT6 on proliferation and apoptosis, the expression of ERK1/2 and p-ERK1/2 were determined following overexpression or knockdown of SIRT6. Additionally, the role of the ERK1/2 signal pathway in the effects of SIRT6 was explored by measuring the effects of the ERK1/2 inhibitor U0126 on the proliferation of SIRT6-overexpressing Huh-7 cells. It was demonstrated that the phosphorylation of ERK1/2 was significantly increased in the SIRT6 overexpression group (P<0.01; Fig. 4G and I), and decreased in the siSIRT6 group compared with the control and NC groups (P<0.01; Fig. 4H and J). Additionally, it was revealed that the proliferation of Huh-7 cells was significantly decreased in the SIRT6 + U0126 group compared with the SIRT6 group (P<0.05), and was significantly decreased in the U0126 group compared with the control (P<0.05; Fig. 5). Collectively, the results suggested that the ERK1/2 signal pathway was activated by overexpression of SIRT6 and downregulated by knockdown of SIRT6, and that the ERK1/2 signal pathway may be involved in the SIRT6-mediated regulation of HCC cell proliferation.