Paired HCC tissues (n=37) and matched non-tumor samples were obtained from a tissue bank of samples collected from patients that underwent surgical treatment between May 2010 and March 2016 at The First Affiliated Hospital of Soochow University (Suzhou, China). Patient characteristics are listed in Table I. The clinicopathological significance of ISG15 levels in patients with HCC was studied according to methods described in the previous studies (20–22). Following surgical resection, the tissue samples were immediately snap frozen in liquid nitrogen and stored at −80°C. Patients that had received radiotherapy and/or immunotherapy prior to surgical treatment were excluded from the current study. Written informed consent was obtained from all patients. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by The First Affiliated Hospital of Soochow University.

ISG15 protein expression in 37 paired paraffin-embedded tumor and adjacent normal tissues was detected using an immunoperoxidase method. In brief, following deparaffinization of samples, antigen retrieval was performed in a sodium citrate solution (pH 6.0). Tumor and adjacent normal tissues were cut into 3-µm-thick sections, and the sections were blocked with normal goat serum at 37°C for 60 min and incubated with rabbit anti-ISG15 antibody (1:100; cat. no. ab131119; Abcam) overnight at 4°C. Subsequently, sections were washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:1,000; cat. no. ab6721; Abcam) at room temperature for 1 h. Finally, 3,3′-diaminobenzidine tetrahydrochloride (DAB; ZSGB-BIO; OriGene Technologies, Inc.) was used as a substrate to visualize the bound antibody for 30 min at 20°C, following which the sections were counterstained with hematoxylin for 10 min at 20°C, dehydrated with an ascending series of ethanol concentrations and mounted with neutral resin. The samples observed with a light microscope (magnification, ×400).

Four HCC cell lines Hep3B, Huh7, MHCC97-H and MHCC97-L (23,24) were purchased from the Type Culture Collection of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO₂.

For ISG15 knockdown, small interfering RNA (siRNA) targeting ISG15 (referred to as siISG15) was purchased from Shanghai GenePharma Co., Ltd., and its sequence was as follows: 5′-TGAGCACCGTGTTCATGAATCTGCG-3′. A negative control siRNA (siNC) was also obtained, as follows: 5′-UCUCCGAACGUGUCACGUTT-3′. HCC cells were transfected with siISG15 or siNC using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocols. For overexpression of ISG15, human ISG15 was cloned into a pcDNA3.1 expression vector. The ISG15 expression vector or control vector (NC) was then transfected into the cells using Lipofectamine 2000, according to the manufacturer's protocol. In order to confirm the knockdown and overexpression, ISG15 mRNA and protein expression levels were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively.

The expression of ISG15 was evaluated by RT-qPCR. Briefly, cells were seeded in 6-well plates at a density of 5×10⁵ cells/well, cultured overnight and then subjected to transfection for 48 h. Total RNA was extracted from the clinical specimens and cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The total RNA was then reverse transcribed into cDNA using the TransCript One-step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech Co., Ltd.). ISG15 expression in cells was detected by qPCR using SYBR Green I chemistry (TransStart Top Green qPCR SuperMix; TransGen Biotech Co., Ltd.). The cycling parameters defined as follows: Initial cycling for 5 min at 95°C, followed by 40 cycles at 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. The primers used for the amplification of the indicated genes were designed using the Primer Express Software (Applied Biosystems; Thermo Fisher Scientific, Inc.), and were as follows: RPN2 forward, 5′-GGACCTGACGGTGAAGATG-3′, and reverse, 5′-TGGTGTTCCGAAGTTGGTCA-3′ (product, 142 bp); GAPDH forward, 5′-ACCCAGAAGACTGTGGATGG-3′, and reverse, 5′-TCAGCTCAGGGATGACCTTG-3′ (product, 124 bp). The relative expression level was calculated according to the 2^−ΔΔCq method, where ΔCq=Cq (gene of interest)-Cq (housekeeping gene) (25). GAPDH served as an internal control. All RT-qPCR reactions were performed in triplicate.

Cells were seeded at a density of 5,000 cells/well in 96-well plates at 24 h after transfection and then treated with norcantharidin (CAS: 5442-12-6; 40 µg/ml; Shanghai Yuanye Biotechnology Co., Ltd.) or DMSO. Cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) assay at 24, 48 and 72 h following seeding. Absorbance was determined at 450 nm using a microplate spectrophotometer.

Colony formation assays were used to assess the clonogenic ability of HCC cells. After 24 h transfection and then 48 h treatment with norcantharidin, cells (2×10³/well) were trypsinized into single-cell suspensions and seeded in 6-well dishes. Subsequently, cells were maintained in DMEM supplemented with 10% FBS for ~2 weeks. Subsequently, visible colonies were fixed in 4% paraformaldehyde for 4 h at 37°C and stained with 0.5% crystal violet for 2 h at 37°C (Beyotime Institute of Biotechnology). Colony numbers were counted under a light microscope.

Cells were harvested, washed with ice-cold PBS and stained with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kits (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. Cell apoptosis was measured using a flow cytometer, and the results were analyzed with FlowJo software, version 7.6.1 (FlowJo LLC).

HCC cells were dissociated using the Total Protein Extraction kit (Wuhan Goodbio Technology Co., Ltd.) according to the manufacturer's instructions, and the extracted protein was examined by western blotting. Protein concentrations were assessed using a bicinchoninic acid assay prior to loading. Total protein lysates (40 µg protein/lane) were resolved by SDS-PAGE on 10% gels and then transferred to polyvinyl difluoride membranes (EMD Millipore). Following blocking in TBS containing 0.1% Tween-20 (TBS-T) with 5% nonfat dry milk for 30 min at 37°C, the membranes were washed with TBS-T (4X) and incubated overnight at 4°C with primary antibodies. All the primary antibodies used in this experiment were obtained from Abcam and were as follows: Anti-caspase-9 (1:800; cat. no. ab202068), anti-caspase-3 (1:500; cat. no. ab13847), anti-GAPDH (1:1,000; cat. no. ab5174), anti-B-cell lymphoma 2 (Bcl-2; 1:1,000; cat. no. 196495), anti-Bcl-2-like protein 4 (Bax; 1:1,000; cat. no. ab32503) and anti-ISG15 (1:2,000; cat. no. ab131119). Following extensive washing, the membranes were incubated with polyclonal HRP-conjugated goat anti-rabbit IgG secondary antibody (cat. no. 7074; CST Biological Reagents Co., Ltd.) at a dilution of 1:2,000 for 1 h at 25°C. Immunoreactivity was detected with an enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.), and subsequently visualized using a ChemiDoc XRS imaging system and analysis software (Bio-Rad Laboratories, Inc.). GAPDH served as the internal loading control.

Data from three independent experiments are expressed as the mean ± standard deviation. Statistical analysis was performed using SPSS software, version 19.0 (IBM Corp.). Student's t-test was used to compare the mean values between two groups, while one-way analysis of ANOVA with Dunnett's multiple comparisons test was used to compare the mean values among multiple groups. Pearson's χ² test was conducted for correlation analysis between the clinicopathological features and ISG15 expression of patients with HCC. P<0.05 was considered to indicate a statistically significant difference.

To identify the biological role of ISG15 in HCC, the expression of ISG15 was detected in 37 paired HCC and normal tissues. As presented in Fig. 1A, the expression of ISG5 was significantly increased in HCC tissues, as compared with that in normal tissues (P<0.001). The protein expression of ISG15 was examined by IHC analysis, and representative images are presented in Fig. 1B. It was observed that ISG15 was evidently overexpressed in HCC tissues as compared with the adjacent non-tumor tissues (Fig. 1B). Additionally, the mRNA levels of ISG15 in a number of HCC cell lines, including Hep3B, Huh7, MHCC97-H and MHCC97-L were evaluated by RT-qPCR. Among the HCC cell lines assessed, Huh7 cells exhibited the highest expression of ISG15, while Hep3B cells exhibited the lowest expression of ISG15. For this reason, downregulation of ISG15 was performed in Huh7 cells, whereas overexpression of ISG15 was performed in Hep3B cells.

As described in previous studies (21–23), the clinicopathological significance of ISG15 levels in patients with HCC was further investigated. The 37 patients were divided into two subgroups based on the mean expression value, which included the low (n=15) and high (n=22) ISG15 expression groups. As presented in Table I, ISG15 levels in HCC tissues were positively correlated with the tumor size, TNM stage and tumor differentiation (P<0.05). Taken together, these results indicated that ISG15 may function as an oncogene in HCC.

To identify the effect of ISG15 on the sensitivity of HCC to norcantharidin, Huh7 cells were transfected with siISG15 and then treated with norcantharidin. The expression of ISG15 in Huh7 cells was assessed by RT-qPCR and western blot analysis, confirming that downregulation was successfully established in the siISG15 group (Fig. 2A and B). Next, cell proliferation and apoptosis were measured by CCK-8, colony formation and Annexin V/PI staining assays. The results revealed that cell proliferation and colony formation were significantly decreased in cells treated with siISG15 and/or norcantharidin, with the combination of siISG15 and norcantharidin leading to the lowest cell viability and number of colonies (P<0.01; Fig. 3C and D). Annexin V/PI staining demonstrated that treatment with siISG15 and/or norcantharidin significantly induced apoptosis in Huh7 cells (P<0.01; Fig. 3E). Combined treatment with siISG15 and norcantharidin significantly promoted apoptosis when compared with the siISG15 or norcantharidin single treatments (P<0.01; Fig. 3E). Taken together, these results suggested that downregulation of ISG15 may promote the antitumor effect of norcantharidin on Huh7 cells.

To investigated whether norcantharidin reverses the tumor-promoting effects of ISG15 in HCC cells, Hep3B cells were transfected with an ISG15 overexpression plasmid and then treated with norcantharidin. The transfection efficiency was measured using RT-qPCR and western blot analysis. It was revealed that ISG15 was significantly overexpressed in Hep3B cells transfected with the plasmid, as compared with the control group (P<0.001; Fig. 3A and B). Cell proliferation and apoptosis were then analyzed. As presented in Fig. 3C and D, ISG15 overexpression alone significantly increased the cell proliferation and colony formation ability compared with the control group (P<0.001). By contrast, treatment with norcantharidin resulted in marked reduction in cell proliferation and colony formation compared with the control (P<0.001). However, the combination treatment significantly reduced the norcantharidin-induced inhibition of cell proliferation and colony formation, suggesting that ISG15 overexpression may reverse the antiproliferative effect of norcantharidin (P<0.001; Fig. 3C and D). Furthermore, the results of Annexin V/PI staining analysis revealed that norcantharidin induced marked cell apoptosis (P<0.001; Fig. 3E), while ISG15 overexpression reversed the effect of norcantharidin on the apoptosis of Hep3B cells (P<0.001; Fig. 3E). The results suggest that ISG15 overexpression can reverse the inhibitory effects of norcantharidin on HCC cells.

The expression levels of apoptosis-associated proteins in HCC cells were detected by western blot analysis. As presented in Fig. 4A, treatment with norcantharidin alone significantly increased the protein expression levels of Bax, caspase-3 and caspase-9, and decreased Bcl-2 expression (P<0.01). Downregulation of ISG15 markedly aggravated the effects of norcantharidin on expression of apoptosis-associated proteins in HCC cells. Furthermore, overexpression of ISG15 was observed to significantly inhibit the expression levels of Bax, caspase-3 and caspase-9, and increase Bcl-2 expression, as compared with the control group (P<0.05; Fig. 4B). Upon combined treatment with the plasmid and norcantharidin, ISG15 overexpression was found to markedly reverse the effect of norcantharidin on the protein expression. These findings suggest that proteins associated with apoptosis were regulated by ISG15 and norcantharidin treatment.