Data from 100 HCC and 35 normal tissues were downloaded from The Cancer Genome Atlas (TCGA, cancergenome.nih.gov) and analyzed via gene set enrichment analysis (GSEA). GSEA was performed using the GSEA software, version 2.0.1, obtained from the Broad Institute (www.broad.mit.edu/gsea) as previously described (17,18). Ethical approval for the present study was provided by the Independent Ethics Committee of Hubei University of Chinese Medicine, Ministry of Education Key Laboratory of Traditional Chinese Medicine Resources and Compounds (Hubei, China).

HCC cell lines (HepG2, Hep3B and SMMC-7721), and the human hepatic cell line (L02) were obtained from JRDun Biotechnology Co., Ltd. (Shanghai, China), cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in an atmosphere containing 5% CO₂ for 48 h. The shRNA target position 538–556 (5′-CCATGAGAAACAGCGTCAA-3′) on human ET-1 mRNA was cloned into PLKO.1-EGFP lentivirus vectors (Addgene, Inc., Cambridge, MA, USA). Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transfection of the constructs according to the manufacturer's protocol. Empty lentivirus vectors were used as a negative control (NC). The SMMC-7721 cells were analyzed 48 h post-transfection.

Total RNA was isolated from the HCC cell lines including HepG2, Hep3B and SMMC-7721 using TRIzol reagent at −80°C. cDNA was synthesized using an M-MLV RT reagent kit (Thermo Fisher Scientific, Inc.). DNase I treatment was used to remove genomic DNA. The reaction mixture contained RNA-Primer mix (12 µl), 5X RT Reaction buffer (5 µl), 25 mM dNTPs (1 µl), 25 U/µl RNase inhibitor (1 µl), 200 U/µl M-MLV RTase (1 µl), Oligo (dt)₁₈ (1 µl) and DNase-free ddH₂O (4 µl). PCR amplification was performed using Maxima SYBR Green/ROX qPCR Master Mix (K0223; Finnzymes; Thermo Fisher Scientific, Inc.) on the ABI (ABI-7500) qPCR platform (Invitrogen; Thermo Fisher Scientific, Inc.). The reaction mixture contained SYBRGreen Mix (12.5 µl), forward primer (0.5 µl), reverse primer (0.5 µl), ddH₂O (9.5 µl) and cDNA (2 µl). GAPDH RNA was used as an internal control to normalize the expression of ET-1. The PCR primers for ET-1 and GAPDH are: ET-1, forward: 5′-GCCTGTCTGAAGCCATAG-3′, reverse: 5′-GCTGAGAGGTCCATTGTC-3′; and GAPDH, forward: 5′-CACCCACTCCTCCACCTTTG-3′, reverse: 5′-CCACCACCCTGTTGCTGTAG-3′. The PCR cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 45 sec, and a final extension step of 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and 60°C for 15 sec. Relative ET-1expression levels were quantified using the 2^−ΔΔCq method (19). The experiment was repeated three times.

Isolated proteins were isolated using RIPA buffer containing 50 mM Tris-HCl, (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, phosphatase and protease inhibitor cocktail (Merck Millipore, Darmstadt, Germany) at 4°C for 20 min, followed by centrifugation at 12,000 × g for 1 min at 25°C. Equivalent quantities (50 µg) of protein lysates were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes (Merck Millipore). Membranes were blocked in fat-free milk overnight at 4°C and subsequently incubated with primary antibodies: anti-ET-1 (cat. no. ab117757; Abcam, Cambridge, UK; 1:1,000) and anti-GAPDH (cat. no. ab9485; Abcam; 1:1,500) at 4°C overnight. Membranes were subsequently washed three times with TBS with Tween-20 (Amresco, LLC, Solon, OH, USA) for 5 min and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (cat. no. A0208; Beyotime Institute of Biotechnology; 1:1,000) at 37°C for 1 h. Signals were detected using an electrochemiluminescence system (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and signal intensity was determined using ImageJ software version 1.46 (National Institutes of Health, Bethesda, MD, USA).

SMMC-7721 cells (3×10³ cells/well) transfected with or without ET-1 shRNA were seeded in a 96-well plate and incubated at 37°C for 24, 48 and 72 h. Cell proliferation was assessed every 24 h throughout the experiment using a Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Absorbance was measured using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm.

SMMC-7721 cells (1×10⁵ cells/well) transfected with or without ET-1 shRNA were seeded into 12-well plates and incubated at 37°C. For the cell cycle assay, cells were removed at specified time points, washed twice with phosphate-buffered saline (PBS), fixed in cold ethanol for 30 min, and then incubated with 100 µg/ml propidium iodide (PI) and 0.5 µg/ml RNase A (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 30 min at 37°C. For the apoptosis assay, cells were stained with 195 µl fluorescent probe-labeled Annexin V-APC and 5 µl PI staining kit (BD Biosciences, Franklin Lakes, NJ, USA). The cell cycle and apoptosis were analyzed using a flow cytometer (BD Accuri C6 version 1.0.264.21 software; BD Biosciences).

SMMC-7721 cells transfected with or without ET-1 shRNA were resuspended in 100 µl RPMI-1,640 medium and placed into the upper chamber of a Transwell support and incubated for 24 h at room temperature. Migratory cells were fixed with 4% polyoxymethylene (China National Pharmaceutical Group Corp., Beijing, China) and stained with 0.2% crystal violet. Evaluation of migrated cells was performed using a biological fluorescence microscope (CX41RF; Olympus Corporation, Tokyo, Japan).

For the in vivo experiment, five-week-old male nude mice (n=12; 15–18 g) were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd., (Shanghai, China). A total of 1×10⁷ SMMC-7721 cells stably expressing shRNA or NC were injected into the right flanks of nude mice to induce tumorigenesis. Mice were housed in the animal facility at a temperature of 25°C and humidity of 60–70% and subjected to a 12-h light-dark cycle with ad libitum access to food and water. The size of tumors was determined at six weeks post-injection as previously described (20). At 42 days post-injection, mice were sacrificed via anesthesia with 3% sodium pentobarbital (40 mg/kg; Sigma-Aldrich; Merck Millipore) via intraperitoneal injection and cervical dislocation was performed. Ethical approval was provided by the Independent Ethics Committee of Hubei University of Chinese Medicine, Ministry of Education Key Laboratory of Traditional Chinese Medicine Resources and Compounds.

Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons among groups were made using two-tailed Student's t-tests. P<0.05 was considered to indicate a statistically significant difference.

To identify the functions of ET-1 in HCC, ET-1 expression was assessed using bioinformatics analysis and RT-qPCR. The results demonstrated that ET-1 expression was significantly increased in the tissues of patients with HCC compared with normal hepatic tissues in the TCGA dataset (P<0.001; Fig. 1A). The expression of ET-1 in HCC cell lines including L02, HepG2, Hep3B and SMMC-7721 were also examined, with a significant upregulation of ET-1 observed in all HCC cells compared with the normal hepatic L02 cells, most notably in the SMMC-7721 cell line (P<0.01 for HepG2 cells, P<0.001 for Hep3B and SMMC-7721 cells; Fig. 1B). As a result, SMMC-7721 cells were used for all subsequent experiments. These results suggest that ET-1 may function as a tumor promoter in HCC.

To investigate the roles of ET-1 in HCC development and progress, ET-1 shRNA and NC shRNA were transfected into SMMC-7721 cells. ET-1 mRNA expression was significantly reduced by 47.5% in SMMC-7721 cells transfected with ET-1 shRNA compared with the NC group (P<0.01; Fig. 2A). ET-1 protein level was markedly decreased in the ET-1 shRNA group compared with the NC and control groups (Fig. 2B).

The carcinogenesis of ET-1 in SMMC-7721 cells was investigated. CCK-8 was used to measure the proliferation of SMMC-7721 cells transfected with ET-1 shRNA in vitro. It was demonstrated that downregulation of ET-1 significantly inhibited the proliferation of SMMC-7721 cells compared with the NC group (P<0.05 for 48 h and P<0.01 for 72 h; Fig. 2C). The effect of ET-1 on tumor growth in vivo was subsequently examined. As illustrated in Fig. 2D, the tumor grew markedly faster in mice injected with SMMC-7721 cells transfected with ET-1 shRNA compared with those transfected with NC shRNA. These data indicate that ET-1 downregulation is able to suppress tumor growth both in vitro and in vivo.

At 48 h post-transfection, flow cytometry was performed to analyze the cell cycle and apoptosis of SMMC-7721 cells. Cell cycle analysis revealed that ET-1 downregulation significantly increased the percentage of cells in G1 phase (P<0.001) and significantly decreased the percentage of cells in S phase (P<0.001), suggesting that downregulation of ET-1 resulted in cell cycle arrest in G1 phase (Fig. 3A and B). The effect of ET-1 on cell apoptosis was assessed and the results of an Annexin V-APC/PI staining assay revealed that ET-1 downregulation also significantly induced cell apoptosis of SMMC-7721 cells by 6.73-fold compared with the NC group (P<0.001; Fig. 3C and D).

To further characterize the function of ET-1 in HCC, a GSEA was performed. mRNA profiling microarray data of HCC tissues were retrieved from TCGA dataset. Based on the median expression level (0.018) of ET-1, HCC tissues were divided into ET-1 high (>0.018) and ET-1 low groups (<0.018). A ranked gene list was generated by comparing the mRNA microarray data of the ET-1 high group with the ET-1 low group (ET-1 high vs. low). The inverse correlation between the expression levels of ET-1 and multiple genes in the cell apoptosis pathway indicated that ET-1 may positively regulate this pathway (Fig. 4A).

The expression of cell apoptosis-associated genes was evaluated at mRNA and protein levels in SMMC-7721 cells. The results demonstrated that the ratio of Bax/Bcl-2 was significantly increased in SMMC-7721 cells transfected with ET-1 shRNA at mRNA and protein levels, compared with the NC group (P<0.001; Fig. 4B and C).

To investigate the effect of ET-1 on migration of SMMC-7721 cells, a Transwell assay was performed to evaluate migratory ability. ET-1 downregulation significantly inhibited the migration of SMMC-7721 cells in the ET-1 shRNA group compared with the NC group (P<0.001; Fig. 5A and B). The expression levels of MMP-2 and MMP-9 in SMMC-7721 cells were also evaluated and the mRNA levels of MMP-2 and MMP-9 were significantly inhibited in the ET-1 shRNA group compared with the NC group (P<0.001 for MMP-2, P<0.001 for MMP-9; Fig. 5C). Similarly, the protein levels of MMP2 and MMP-9 were markedly inhibited by ET-1 downregulation compared with the NC group (Fig. 5D).