Small molecule inhibitors were as follows: MG-132 (cat. no. 474790; EMD Millipore), cycloheximide (CHX; cat. no. C7698, Sigma-Aldrich; Merck KGaA) and leptomycin B (LMB; cat. no. HY-16909; MedChemExpress). TGF-βI was purchased from PeproTech, Inc. (cat. no. 100-21-2). Briefly, MG-132 was used at a concentration of 10 µM for 12 h at 37°C; LMB was used at a concentration of 50 ng/ml for 12 h at 37°C; TGF-βI was used at a concentration of 20 ng/ml for 24 h at 37°C in Huh7 cells. All other chemical reagents were obtained from Sigma-Aldrich (Merck KGaA), unless otherwise indicated.

This study employed 293T cells and two hepatocellular carcinoma cell lines, Huh7 and PLC/PRF/5. All cell lines were obtained from the CellCookBiotech Co., Ltd. Mycoplasma testing was performed for all the cell lines used, which were authenticated by short tandem repeat analysis. Cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C and 5% CO₂. Cells were digested and passaged as previously described (17), passage 3–7 (early passages) were used for assays.

Cells were trypsinized and pelleted via centrifugation at a speed of 400 × g for 5 min at room temperature. After washing twice in 1X PBS, Huh7 cells were resuspended in serum-free DMEM at a density of 5×10⁵ cells/ml, and 200 µl cell suspension was seeded onto the upper chambers of the Transwell inserts (pore size, 8 µm; cat. no. 3422; Corning). DMEM containing 10% FBS was added to the lower chamber, FBS served as a chemoattractant. After incubation at 37°C for 20 h, the non-migrated cells were gently removed with a cotton swab. Migrated cells located on the lower side of the chamber were fixed with 4% paraformaldehyde for 20 min at room temperature prior to crystal violet (cat. no. C0121; Beyotime Institute of Biotechnology) staining for 1 h at room temperature. The number of migrated cells was examined via light microscopic observation at ×200 magnification (DMi8; Leica Microsystems, Inc.).

The cell proliferative rate was determined using a Cell Counting Kit-8 (CCK-8) assay (cat. no. CK04; Dojindo Molecular Technologies, Inc.), according to the manufacturer's protocol. In brief, Huh7 cells were seeded in five replicates in a 96-well plate at a density of 1,000 cells and cultured with 100 µl DMEM containing 10% FBS per well. For measurement, 10 µl CCK-8 solution was added to each well, and the cells were incubated for another 4 h at 37°C. Viable cells were counted every day for a period of 5 days by reading the absorbance at 450 nm with a plate reader (ELx800; BioTek Instruments, Inc.).

In brief, 5×10⁵ Huh7 cells were harvested in fresh medium. Samples were washed in 1X PBS, and then fixed in ice-cold 70% ethanol at −20°C overnight. Fixed cells were washed with cold 1X PBS and stained with FxCycle™ PI/RNase staining solution (cat. no. F10797; Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min at room temperature in the dark. Cells were then analyzed using a BD LSR II™ flow cytometry (BD Biosciences), and data were analyzed with FlowJo software (version 10; FlowJo LLC).

Total RNA was isolated from 293T or Huh7 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA (1 µg) was reverse transcribed at 42°C for 1 h into cDNA using the GoScript™ Reverse Transcription system (cat. no. A5002; Promega Corporation) according to the manufacturer's protocol. PCR was performed with Platinum SYBR-Green qPCR SuperMix-UDG (Invitrogen; Thermo Fisher Scientific, Inc.) with a LightCycler 480 PCR platform (Roche Diagnostics). The thermocycling conditions were as follows: Initial denaturation for 10 min at 95°C, 40 cycles of denaturation for 15 sec at 95°C, annealing for 40 sec at 55°C and elongation for 20 sec at 72°C, followed by final extension for 3 min at 72°C. Specific primers were as follows: GTSE1 forward, 5′-CAGGGGACGTGAACATGGATG-3′ and reverse, 5′-ATGTCCAAAGGGTCCGAAGAA-3′; SNAIL1 forward, 5′-TCGGAAGCCTAACTACAGCGA-3′ and reverse, 5′-AGATGAGCATTGGCAGCGAG-3′; and GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. Relative expression levels were calculated using the 2^−ΔΔCq method (18) following normalization to the expression of GAPDH.

Cells were lysed in NETN buffer (20 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific, Inc.). The lysate protein concentration was measured using the BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). After normalization to equal amounts, 10 µg each protein sample was separated via 10% SDS-PAGE, transferred to PVDF membranes and blocked with 5% non-fat milk diluted in 1X PBS supplemented with 0.5% Tween-20 (PBST) at room temperature for 1 h. Then, the membranes were probed with the following primary antibodies at 4°C overnight: GTSE1 (cat. no. A302-425A; Bethyl Laboratories, Inc.; 1:1,000), SNAIL1 (cat. no. 3879; Cell Signaling Technology, Inc.; 1:1,000), Tubulin (cat. no. 66031-1-Ig; ProteinTech Group, Inc.; 1:5,000), Lamin B1 (cat. no. 66095-1-Ig; ProteinTech Group, Inc.; 1:1,000), anti-Flag tag (cat. no. F3165; Sigma-Aldrich; Merck KGaA; 1:3,000) and anti-human influenza hemagglutinin epitope (HA) tag (the amino acid sequence is YPYDVPDYA; cat. no. 3724; Cell Signaling Technology, Inc.; 1:1,000). The blots were then incubated with species-specific HRP-conjugated secondary antibodies (cat. no. W4011 for rabbit and cat. no. W4021 for mouse-originated primary antibodies; Promega Corporation; 1:5,000) at room temperature for 1 h, and the immunoreactive bands were visualized using an ECL reagent (cat. no. 6883; Cell Signaling Technology, Inc.). The primary antibodies were diluted in Primary Antibody Dilution Buffer (cat. no. P0023A; Beyotime Institute of Biotechnology), and the secondary antibodies were diluted in 1X PBST. Tubulin or Lamin B1 was used as a loading control. Semi-quantification of band densitometry was measured with ImageJ software (version 1.47; National Institutes of Health).

DNA fragments containing the coding sequence (CDS) of full-length human GTSE1 and SNAIL1 were amplified via nested PCR from a cDNA library of 293T cells using the PrimeSTAR^® Max DNA Polymerase (cat. no. R045A; Takara Bio, Inc.). The thermocycling conditions were as follows: Initial denaturation for 3 min at 98°C, followed by 35 cycles of denaturation for 15 sec at 98°C, annealing for 5 sec at 55°C and elongation for 30 sec at 72°C, followed by final extension for 5 min at 72°C. The outer primer pairs used were as follows: GTSE1-outer-forward, 5′-GTTTAAATCCGTGCCGGAGG-3′; GTSE1-outer-reverse, 5′-AGGGCTGTTCTTTCAAGGCA-3′; and SNAIL1-outer-forward, 5′-AGTGGTTCTTCTGCGCTACT-3′; SNAIL1-outer-reverse, 5′-AGGCTGAAATAGCTGCCTGG-3′. The product of the first PCR was used as the template for the second PCR during which the sequences encoding Flag tag (5′-GATTACAAGGATGACGACGATAAG-3′) or HA tag (5′-TACCCATACGATGTTCCAGATTACGCT-3′) were added after the start codon to the 5′ end of the CDS region of the indicated protein. The product of the second PCR was then digested and ligated into the KpnI and EcoRI sites of the pcDNA3.1(+) vector (Invitrogen; Thermo Fisher Scientific, Inc.). Correct constructs were all confirmed via Sanger sequencing with the CMV-forward primer 5′-CGCAAATGGGCGGTAGGCGTG-3′.

Huh7 or PLC/PRF/5 cells were transfected with plasmids encoding GTSE1 and/or SNAIL1, and negative control pcDNA3.1(+) plasmid using ViaFect™ transfection reagent (Promega Corporation) for 6 h at 37°C in a cell incubator following the manufacturer's protocol. For transfection in 6-well plates, a total of 2 µg plasmid was used for each well. At 36 h post-transfection, the transfection efficiency was detected using WB.

A total of two targeting GTSE1 small interfering (si)RNA duplexes (siGTSE1#1, 5′-GGATGTTCTCCCTGACAAA-3′; siGTSE1#2, 5′-GCCTACTCCTACAAATCAA-3′) and the negative control (siN0000001) were obtained from Guangzhou RiboBio Co., Ltd. Huh7 cells were transfected with 100 nM siRNA using Lipofectamine^® RNAiMax (Invitrogen; Thermo Fisher Scientific, Inc.) for 24 h at 37°C in a cell incubator, according to the manufacturer's protocol. At 72 h post-transfection, RNA interference was confirmed via RT-qPCR or WB.

Cells (70% confluence) were plated on chamber slides and fixed with 4% paraformaldehyde at room temperature for 5 min. After fixation, cells were permeabilized with 0.1% Triton X-100 for 5 min. Then, cells were blocked with 10% FBS for 20 min at room temperature and incubated with the following primary antibodies at 4°C overnight: Anti-Flag tag (cat. no. F3165; Sigma-Aldrich; Merck KGaA; 1:1,000) and anti-HA tag (cat. no. 3724; Cell Signaling Technology, Inc.; 1:500). The anti-Mouse IgG (H+L) Alexa Fluor^® 488 conjugate (cat. no. A-11001; Invitrogen; Thermo Fisher Scientific, Inc.; 1:200) or anti-Rabbit IgG (H+L) Alexa Fluor^® 594 conjugate secondary antibodies (cat. no. A-11012; Invitrogen; Thermo Fisher Scientific, Inc.; 1:200) were added and incubated in the dark for 60 min at room temperature. Nuclear staining was performed with 50 ng/ml DAPI (cat. no. D21490; Invitrogen; Thermo Fisher Scientific, Inc.) in the dark for 5 min at room temperature. The fluorescence signal was imaged using a Zeiss LSM710 confocal microscope at ×400 magnification (Zeiss AG).

A GTSE1 promoter region from −1,500 to the transcription start site (TSS) was amplified via PCR, which was performed as aforementioned, and inserted into the KpnI and HindIII sites of pGL3-Basic plasmid (Promega Corporation), hereafter named pGL3-GTSE1-PM. In total, three potential Smad binding elements (SBEs) containing the 5 bp CAGAC motif were found in the aforementioned GTSE1 promoter region, locating at −35 to −39, −714 to −718 and −1,117 to −1121, respectively. Accordingly, three GTSE1 promoter constructs, deleting one of the potential SBEs, were prepared via a PCR-based method, and were named pGL3-GTSE1-DEL1, pGL3-GTSE1-DEL2 and pGL3-GTSE1-DEL3.

A total of ~1×10⁵ Huh7 cells were seeded in 12-well plates and cultured for 24 h. Then, 1 µg each of the GTSE1 promoter constructs and 0.05 µg control pRL-TK (Promega Corporation) encoding Renilla luciferase were then co-transfected into cells using ViaFect™ transfection reagent (Promega Corporation) for 6 h at 37°C in a cell incubator, following the manufacturer's procedures. At 30 h post-transfection, quantification of firefly and Renilla luciferase activities of ≥3 independent transfections were measured with the Dual Reporter Assay system (Promega Corporation) using an FB12 luminometer (Titertek-Berthold). The relative luciferase activities were calculated by normalizing the activity of the fluorescent luciferase with that of the internal standard Renilla luciferase.

Huh7 cells were transiently transfected with GTSE1 encoding plasmid and the negative control plasmid, or GTSE1 targeting siRNAs and negative control siRNA. At 30 h post-transfection with plasmids or 72 h post-transfection with siRNAs, transfected cells were treated with 10 µg/ml CHX at 37°C for an additional time period, including 0, 0.5, 1 and 2 h. Then, cells were lysed in NETN buffer, and the SNAIL1 protein turnover was detected via WB, performed as aforementioned, using antibodies against GTSE1, SNAIL1, HA tag and Tubulin. Tubulin was used as a loading control.

Nuclear and cytoplasmic extracts of Huh7 cells were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce; Thermo Fisher Scientific, Inc.) according to the supplier's protocol.

SPSS software version 20.0 (IBM Corp.) and GraphPad Prism 6 software (GraphPad Software, Inc.) were used to perform statistical analyses. Statistical differences between two groups were determined using an unpaired Student's t-test, whereas statistical differences between multiple groups were determined using a one-way ANOVA, followed by a Tukey's post hoc test. Each experiment was performed three times in triplicate. Unless otherwise indicated, all error bars indicate SD. All statistical tests were two-sided, and P<0.05 was considered to indicate a statistically significant difference.

Our previous study reported that GTSE1 was involved in regulating the expression level of SNAIL1 protein and promoted the metastatic ability of HCC cells via EMT process (13). However, the exact mechanism via which GTSE1 regulates SNAIL1 expression is unknown. The current study first examined the relationship between GTSE1 and SNAIL1 expression in Huh7 and PLC/PRF/5 cells. Transfection of plasmids encoding HA-GTSE1 caused notable overexpression of GTSE1, which reduced the protein expression level of SNAIL1 in HCC cells (Figs. 1A and S1A). By contrast, knockdown of GTSE1 expression by two independent siRNAs, which notably reduced the protein expression of GTSE1, enhanced the expression of SNAIL1 in HCC cells (Figs. 1B and S1B). These results indicated that GTSE1 negatively regulates protein expression level of SNAIL1 in HCC cells.

Further investigations revealed that GTSE1 induced a decrease in SNAIL1 expression in a concentration-dependent manner, and the addition of the proteasome inhibitor MG-132 reversed the reduction in SNAIL1 expression (Fig. 2A). A CHX chase experiment was then conducted, and it was found that GTSE1 overexpression significantly promoted the degradation rate of SNAIL1 (Fig. 2B), while SNAIL1 protein turnover was delayed after GTSE1 knockdown in HCC cells (Fig. 2C). These results suggested that GTSE1 promoted the degradation of SNAIL1 protein via a proteasome-dependent pathway.

To determine whether GTSE1 expression influenced the subcellular localization of SNAIL1, HA-GTSE1 and Flag-SNAIL1 were co-expressed in HCC cells and an immunofluorescence assay was performed. As a major readout for this assay, exogenous Flag-SNAIL1 overexpression was firstly confirmed via WB in both Huh7 and PLC/PRF/5 cells (Fig. S2). The results demonstrated that Flag-SNAIL1 was mainly localized in the nucleus of HCC cells expressing Flag-SNAIL1 only, while its expression was detected in both the cytoplasm and the nucleus when HA-GTSE1 was co-expressed with Flag-SNAIL1 (Figs. 3A and B and S3A). Subcellular fractionation also demonstrated that GTSE1 overexpression enhanced the cytoplasmic localization of SNAIL1 (Fig. 3C). When the nuclear export inhibitor LMB was added, cytoplasmic retention of SNAIL1 was reduced, and Flag-SNAIL1 again displayed nuclear localization even under GTSE1 overexpression (Figs. 3D and E and S3B). These results indicated that GTSE1 induced the nuclear export of SNAIL1 in HCC cells.

TGF-βI induces the expression of GTSE1 via SBEs in the promoter region. TGF-β/Smad signaling is able to induce EMT via upregulation of SNAIL1 expression in various human cancer types, including HCC (19). To determine whether GTSE1 was involved in TGF-β signaling-mediated regulation of SNAIL1 expression, the expression of endogenous GTSE1 and SNAIL1 was first detected in the presence of TGF-βI, a potent agonist of TGF-β/Smad signaling. Interestingly, an increasing expression level of GTSE1 but a decreasing expression level of SNAIL1 was observed after 24 h of treatment with a steadily growing concentration of TGF-βI (Fig. 4A). Transcriptionally, TGF-βI treatment induced the upregulation of only GTSE1 mRNA expression levels and affect not SNAIL1 mRNA expression levels in Huh7 cells (Fig. 4B).

TGF-βI actives the transcription of gene expression via the binding of the downstream Smad transcription factors to SBE, the 5 bp CAGAC motif, in the promoter region of specific target genes (20). Therefore, the current study analyzed the sequence of the GTSE1 promoter region and found three potential SBEs located at the region from −1,500 to the TSS. Accordingly, luciferase reporter plasmids of GTSE1 promoter were created containing different SBE deletions as indicated in Fig. 4C. Under unstimulated conditions, reporter plasmids with the deletion of either SBE (GTSE1-DEL1, GTSE1-DEL2 or GTSE1-DEL3) showed comparable, but slightly lower transcriptional activity compared with the control plasmid (GTSE1-PM). After 24 h of TGF-βI treatment, transcriptional activity of the GTSE1-PM and GTSE1-DEL3 plasmids increased nearly 5-fold compared with the unstimulated control. However, the transcriptional activity of either GTSE1-DEL1 or GTSE1-DEL2 plasmids showed no obvious induction even after TGF-βI treatment compared with the unstimulated control (Fig. 4D). Moreover, knockdown of GTSE1 via transfection of siRNAs significantly reversed the decrease in SNAIL1 protein expression after TGF-βI treatment, even though the mRNA level of SNAIL1 was not differentially changed (Fig. 4E and F). These results indicated that TGF-βI treatment could upregulate the transcription of GTSE1 expression by transactivating the SBEs in GTSE1 promoter, and that the induced GTSE1 expression further promoted the degradation of SNAIL1 protein.

In order to examine the biological processes altered by GTSE1 expression, the present study measured the cell proliferation rate, cell cycle distribution and migratory ability of Huh7 cells after GTSE1 overexpression or knockdown with or without TGF-βI treatment. Cell proliferation and cell cycle were not significantly altered by TGF-βI treatment after GTSE1 knockdown in Huh7 cells (Fig. S4A and B). However, TGF-βI treatment caused an increase in the migrated cell number of Huh7 cells in the siNC group under TGF-βI treatment conditions, regardless of the decrease of SNAIL1 expression, and there was no significant difference in the migration of the cells in the siGTSE1 group under TGF-βI conditions compared with the control (Fig. 5A). Consistently, GTSE1 overexpression enhanced the migratory ability in Huh7 cells, despite the fact that increased SNAIL1 protein turnover was induced (Fig. 5B). There results suggested that the TGF-βI-enhanced migratory ability was mainly due to the increased expression of GTSE1, but not the expression of SNAIL1, in Huh7 cells.