MHCC97H and L-02 cell lines were provided by the Central Laboratory of Shanghai First People's Hospital (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in 5% CO₂ at 37°C. MHCC97H cells were treated with two drugs, thapsigargin (TG; 5 µmol/l; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and tunicamycin (TU; 5 µg/ml; Beijing Solarbio Science & Technology Co., Ltd.) for 24 h, and DMSO (5 µg/ml; Beijing Solarbio Science & Technology Co., Ltd.) was used as the control.

Transduction was performed using a GenePharma Lentivirus Transduction kit (Shanghai GenePharma Co., Ltd., Shanghai, China) according to the manufacturer's protocol. MHCC97H cells were plated in six-well plates at a density of 2×10⁵ cells/well. Following 24 h at 37°C, lentiviruses carrying four different TRB3/CHOP targeting shRNAs (TRB3-shRNA 778, shRNA 1057, shRNA1185, shRNA1608; CHOP-shRNA 342, shRNA 807, shRNA 814, shRNA 929) and negative control (NC) shRNA (Shanghai GenePharma Co., Ltd.) were added to the cells. The shRNA sequences are shown in Table I. The concentration of shRNA added was 1×10⁸ U/ml (40 µl). The viral titer was 1 ml/well. At the same time, polybrene was added to each well at a final concentration of 6 µg/ml. Following incubation at 37°C for 12 h, the viral supernatant was replaced with DMEM supplemented with 10% FBS. The infected cells were selected using 2 mg/ml puromycin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) following incubation at 37°C for 48 h. Successful infection was confirmed by the expression of green fluorescent protein using an inverted fluorescence microscope (Leica DMI4000 B; Leica Microsystems GmbH, Wetzlar, Germany). After 72 and 96 h at 37°C the total RNA and total proteins were isolated using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) and SDS sample loading buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) respectively to determine the expression levels of target genes and proteins by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The cells were infected every 12 h for 2 days.

A total of 1×10⁴ MHCC97H cells in 100 µl DMEM containing 10% FBS were seeded in a 96-well plate format in 5% CO₂ at 37°C. A total of 1 ml CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was mixed with 10 ml DMEM supplemented with 10% FBS. Following transfection with TRB3-shRNA 1608 at 37°C for 48 h or treatment with cisplatin (4 µg/ml; Sangon Biotech Co., Ltd., Shanghai, China) at 37°C for 24 h, the MHCC97H cells were then transferred into DMEM containing 10% FBS at 37°C for 48, 72 and 96 h, the 100 µl diluted CCK-8 was added to each well and then absorbance at 450 nm following 1 h of cultivation at 37°C condition was determined. The absorbance at the 48 h was used as the standard.

The total mRNA from L-02 and MHCC97H cells was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the Reverse Transcription kit from Takara Biotechnology Co., Ltd. (Dalian, China) according to the manufacturer's protocol. The process of reverse transcription was as follows: 37°C for 15 min, 85°C for 5 sec, then held at 4°C hold. The forward and reverse primers used are presented in Table II. PCR reactions were performed using a FTC3000 Real-Time PCR Detection System (Funglyn Biotech, Inc., Richmond Hill, ON, Canada) with SYBR Premix Ex Taq™ II (Takara Biotechnology Co., Ltd.). The conditions as follows: Pre-denaturation for 3 min at 95°C, followed by 40 cycles of denaturation for 30 sec at 95°C, annealing for 30 sec at 52°C, extension for 30 sec at 72°C and a final cycle extension at 72°C for 10 min. The level of relative gene expression was evaluated using the ΔΔCq method (11), and expression was normalized to GAPDH. All samples were analyzed three times.

The cells were lysed using SDS sample loading buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min on ice. The protein concentration was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of protein extracts (30 µg) were separated electrophoretically on 10 or 15% SDS-PAGE gels and transferred to a polyvinylidene fluoride membrane. The membrane was blocked using 5% w/v bovine serum albumin (Beyotime Institute of Biotechnology), dissolved in TBS with Tween-20 (TBST; 0.1% Tween-20) at room temperature for 1 h and incubated sequentially with primary antibodies for 8 h at 4°C and peroxidase-conjugated secondary antibodies for 1 h at 25°C. The antibodies used were as follows: Rabbit TRB3 polyclonal antibody (1:2,000; cat no. A2346; ABclonal Biotech Co., Ltd., Woburn, MA, USA), rabbit CHOP polyclonal antibody (1:5,000; cat no. A0221; ABclonal Biotech Co., Ltd.), rabbit AKT polyclonal antibody (1:1,000; cat no. A0001; ABclonal Biotech Co., Ltd.), rabbit pAKT-S473 polyclonal antibody (1:1,000; cat no. AP0098; ABclonal Biotech Co., Ltd.), rabbit GAPDH antibody (1:10,000; cat no. AB21612; ABclonal Biotech Co., Ltd.) and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2,000; cat no. SC-2004; Santa Cruz Biotechnology Inc., Dallas, TX, USA). Following washing in TBST, immunobands were visualized using an enhanced chemiluminescence kit (GE Healthcare, Chicago, IL, USA) following exposure to X-ray films. The western blot bands were evaluated using Fusion software (version 8.0; Vilber Lourmat, Marne-la-Vallée, France) to determine the protein expression levels and analyzed using the Quantity One program (version 4.6.9; Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were repeated three times.

Data are presented as the mean ± standard deviation. All statistical analyses were performed using SPSS (version 19.0; IBM Corp., Armonk, NY, USA). Cell proliferation analysis between the experimental and control groups was performed using unpaired t-test. The results from RT-qPCR and western blot analysis were analyzed using one-way analysis of variance. Multiple comparisons between the groups were performed using the Student-Newman-Keuls method. P<0.05 was considered to indicate a statistically significant difference.

The level of TRB3 mRNA expression was previously demonstrated to be altered in various tumor tissues including liver cancer tissue samples from patients and HCC cell lines (HepG2, Hep3B, Huh-7, HLE and HCF) compared with normal cell lines (12). The present study demonstrated an elevated expression level of TRB3 in MHCC97H cells, ~3.8-fold compared with the expression in the L-02 normal hepatic cell line (Fig. 1A). It was revealed that TRB3 mRNA expression level was also altered in the MHCC97H HCC cell line compared to L-02 cells.

To detect the changes in the expression of TRB3, CHOP and AKT under ER stress, MHCC97H cells were treated with two drugs, TG (5 µmol/l) and TU (5 µg/ml), which are known inducers of ER stress. After 24 h, western blotting was performed to analyze the levels of TRB3, CHOP, AKT and pAKT expression. Following treatment with TU or TG, the expression of TRB3 and CHOP was upregulated, whereas pAKT expression was decreased compared to the DMSO control (Fig. 1B). The levels of mRNA expression of TRB3, CHOP and AKT in these cells were also determined by RT-qPCR. The results revealed that the expression levels of CHOP and TRB3 mRNA were increased following treatment with TG or TU. By contrast, the changes in the levels of AKT expression following treatment with TG or TU were not statistically significant (Fig. 1C).

To investigate the function of TRB3 in apoptosis of MHCC97H cells and resistance to chemotherapy, TRB3 was knocked down in the present study by transduction of MHCC97H cells with specific shRNAs. A total of four shRNA sequences targeting various regions of TRB3 mRNA (shRNA-778, shRNA-1507, shRNA-1185 and shRNA-1608) and a single NC shRNA sequence were used, and the knockdown efficiency was subsequently determined. Following lentivirus-mediated transduction for 24 h, the levels of TRB3 expression after 72 and 96 h were determined in the present study. Compared with the NC control-transduced cells, transduction with shRNA-778 or shRNA-1608 was able to inhibit TRB3 expression at 72 and 96 h (Fig. 2A and B). In the present study, the levels of TRB3 protein expression in the transducted cells were also analyzed by western blotting and it was indicated that the level of TRB3 protein expression in shRNA-1608-trasnfected cells was markedly downregulated compared with the expression in the the NC control-transduced cells. (Fig. 2C). Based on these results, the most efficient shRNA sequences for inhibiting TRB3 expression were selected for further experiments.

Following shRNA transfection, CCK-8 assay was used to determine cell survival. The 24 to 96 h cell survival curve revealed that the number of living cells was markedly reduced when TRB3 expression was knocked down compared with the NC-shRNA-transduced cells (Fig. 2D). In addition, to investigate whether the silencing of TRB3 altered the sensitivity of MHCC97H cells to chemotherapeutic agents, TRB3- and NC-shRNA-transduced cells were treated with 4 µg/ml cisplatin for 24 h and cell survival was determined by CCK-8 assay. There was a significantly higher number of surviving cells in the cells transduced with TRB3-shRNA compared with cells transduced with NC-shRNA following treatment with anticancer drug cisplatin for 96 h (Fig. 2E). These results suggested that expression of TRB3 may increase apoptosis of MHCC97H cells and reverse chemotherapy resistance.

The aforementioned ER stress activation experiment confirmed the previous hypothesis that TRB3 expression was upregulated under ER stress conditions and is associated with activation of AKT. For the next part of the present study, CHOP, a well-known molecule involved in ER stress, was knocked down using the same methods for TRB3 knockdown (Figs. 3A-C). Following transduction of CHOP-shRNA, the levels of TRB3 mRNA and protein expression in MHCC97H cells were determined by RT-qPCR and western blotting, respectively. The results indicated that TRB3 expression was significantly downregulated compared with the control group (Fig. 3D and E). These results do not support the hypothesis that ER stress marker CHOP increases the expression level of TRB3.

In the present study, it was also investigated whether increased tumorigenic properties as a result of inhibition of TRB3 expression, relied on increased AKT activation. Therefore, TRB3 expression was knocked down in MHCC97H cells and activation of AKT was investigated. It was indicated that knockdown of TRB3 did not significantly affect the level of AKT expression but significantly increased the level of pAKT compared with the expression in NC shRNA-transducted cells, which suggested that TRB3 was able to prevent AKT activation (Fig. 4A). However, RT-qPCR results revealed that AKT mRNA decreased in TRB3-knocked down MHCC97H cells (Fig. 4B). These results suggested that TRB3 exerted its inhibitory effects on AKT activity at a post-transcriptional level.