The mouse HCC cell line Hepa1-6 was purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium (cat. no. MA0215; Dalian Meilun Biology Technology Co., Ltd.) supplemented with 10% FBS (cat. no. 164210-50; Procell Life Science & Technology Co., Ltd.) and 1% penicillin-streptomycin at 37°C in a 5%-CO₂ incubator.

Cells were treated with a series of LNT dosages (0, 250, 500 and 1,000 µg/ml) for 24 h. LNT was prepared from Lentinus edodes by the Engineering Technological Center of Mushroom Industry and confirmed to be essentially consistent with standards obtained from Jinling Pharmaceutical Co., Ltd. (Nanjing, China).

Cellular growth and proliferation in different groups were monitored via optical microscope and detected with Cell Counting Kit-8 (CCK-8; cat. no. A311; Vazyme Biotech Co., Ltd.) according to the manufacturer's protocol. Briefly, Hepa1-6 cells in the logarithmic growth phase were inoculated into a 96-well microplates at a density of 2×10³ cells per well at 37°C for 12 h. After the cells adhered to the plate wall, the supernatant was discarded and the cells were treated with different concentrations of LNT (0, 250, 500 and 1,000 µg/ml) in a humidified atmosphere containing 5% CO₂ for 12 and 24 h, respectively. Then, 10 µl of CCK-8 reagent was added into each well and incubated for 2 h at 37°C. The absorbance at 450 nm was measured using a microplate reader (Infinite M200 PRO; Tecan Group, Ltd.). Cell viability was calculated as follows: Cell viability (%)=(As-Ab)/(Ac-Ab) ×100%, where Ab, As, and Ac were the values of the blank medium, experimental group, and control group, respectively.

Hepa1-6 cells (6×10⁴) were inoculated into six-well plates, and cultured until adherence, before being treated with different dosages of LNT for 24 h at 37°C. Next, in situ fluorescence staining was performed using an Annexin V-FITC Apoptosis Detection kit (cat. no. C1062L; Beyotime Institute of Biotechnology). Fluorescence was observed under a fluorescence microscope (BX51; Olympus Corporation). FITC and propidium iodide (PI) staining were associated with green and red fluorescence, respectively.

Hepa1-6 cells were inoculated and treated with LNT according to the method that was performed for the apoptosis assay. As a positive control, cells were also treated for 24 h at 37°C with a mixture of 100 µmol/l FeSO₄ and 500 µmol/l H₂O₂. FITC and PI staining were performed using an Annexin V-FITC Apoptosis Detection kit. Fluorescence was measured by flow cytometry (Flowsight; Merck KGaA) and apoptosis was analyzed for each group of cells using the FlowSight software.

For samples in each group, total protein was extracted from cells or tissues by WB and IP cell lysates (cat. no. P0013; Beyotime Institute of Biotechnology) before the BCA Protein Assay kit (cat. no. 23227; Thermo Fisher Scientific, Inc.) was used for quantification. Protein samples (20 µg per well) were subjected to SDS-PAGE on an 8% gel, transferred onto PVDF membranes (cat. no. 3010040001; Roche Diagnostics) after electrophoresis and blocked with NcmBlot blocking buffer (cat. no. P30500; New Cell & Molecular Biotech) for 20 min at room temperature. The membranes were then incubated with primary antibodies (1:1,000) overnight at 4°C, before being incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies for 1 h at room temperature. Finally, an ECL luminescent solution (cat. no. MA0186-1; Dalian Meilun Biology Technology Co., Ltd.) was added and the target protein was detected by exposure using an Omega Lum C Gel Imaging system (Gel Company, Inc.). Quantitative protein analysis was performed using ImageJ software (version: 2.1.0/1.53c; National Institutes of Health).

The following primary antibodies were used: Rabbit EGR1 (cat. no. 55117-1-AP; ProteinTech Group, Inc.), rabbit PTEN (cat. no. AF6351), rabbit phosphorylated (p)-AKT (cat. no. AF0016), rabbit AKT (cat. no. AF6261), mouse Bcl-2 (cat. no. BF9103), rabbit Bax (cat. no. AF0120), rabbit poly (ADP ribose) polymerase 1 (PARP1; cat. no. 13371-1-AP), rabbit heat shock protein 60 (Hsp60; cat. no. AF0184), rabbit proliferating cell nuclear antigen (PCNA; cat. no. AF0239) and mouse β-actin (cat. no. T0022; all from Affinity Biosciences).

The following secondary antibodies were used: Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (cat. no. A-11001), and Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (cat no. A-11008; both from Thermo Fisher Scientific, Inc.).

To induce the overexpression of EGR1, according to the mouse EGR1 mRNA sequence on NCBI (NM-007913.5), the specific primers were designed by Primer Premier 5.0 software (Premier Biosoft International), and 15 bp eukaryotic expression plasmid pCMV-Myc (cat no. 11910ES03; Shanghai Yeasen Biotechnology Co., Ltd.) homologous sequence was added at both ends (Table I). Total RNA was extracted from Hepa1-6 cells by TRIzol Universal (cat. no. DP424; Tiangen Biotech Co., Ltd.) and reverse transcribed into cDNA by PrimeScript™ RT reagent kit (Perfect Real Time) (cat. no. RR037 A; Takara Biotechnology Co., Ltd.). Using this as a template, PCR was carried out with primers (cat. no. 10154; Shanghai Yeasen Biotechnology Co., Ltd.). The reaction procedure is shown in Table II. PCR products were isolated by 1% agarose gel with YeaRed Nucleic Acid Gel Stain (10,000X in DMSO) (cat. no. 10202ES30; Shanghai Yeasen Biotechnology Co., Ltd.) and separated from the gel with a DNA extraction kit (cat no. B518131; Sangon Biotech Co., Ltd.). Subsequently, the pEASY^®-Basic Seamless Cloning and Assembly Kit (cat. no. CU201-02; Beijing Transgene Biotech Co., Ltd.) was applied to clone the amplified cDNA into the linear pCMV-Myc plasmid fragment recovered by EcoRI restriction enzymes (cat. no. R0101; New England Biolabs). The correctly sequenced strains were amplified, and the plasmids were extracted with TIANprep Mini Plasmid kit (cat. no. DP106; Tiangen Biotech Co., Ltd.) for subsequent cell transfection.

Hepa1-6 cells (5×10⁵) were seeded into a six-well plate and cultured in 500 µl serum- and antibiotics-free medium. When the cell density reached about 90%, the cells were transfected with the plasmids. According to the protocol of Hieff Trans™ Liposomal Transfection Reagent (cat no. 40802ES03; Shanghai Yeasen Biotechnology Co., Ltd.), 4 µg of plasmid DNA and 10 µl of Hieff TransTM liposome nucleic acid transfection reagent was mixed with 250 µl of OPTI-MEMI medium and incubated at room temperature (25°C) for 5 min, respectively. Subsequently, the diluted plasmid DNA and liposome nucleic acid transfection reagent were mixed evenly and incubated at room temperature (25°C) for 20 min to form DNA- liposome complex. Then, 500 µl of DNA-liposome complex was added into each plate well and cultured at 37°C in a 5%-CO₂ incubator for 24 h. When necessary, after transfection for 6 h, the cells were cultured with a complete medium for improved transfection activity. Finally, mediums containing gradient concentration of LNT (0, 250, 500, and 1,000 µg/ml) were applied to culture for another 24-h incubation. WB was used to detect the expression level of EGR1 protein.

After 24 h of treatment with gradient concentrations of LNT at 37°C, nuclear and cytoplasmic proteins were extracted and separated using a Nuclear and Cytoplasmic Protein Extraction kit (cat. no. P0028; Beyotime Institute of Biotechnology). Finally, WB was used to detect the expression of EGR1 protein in the two intracellular compartments.

Clean slides were placed into 24-well plates for cell spreading, with six replicate wells set up for each group. Cells at a density of 1×10⁵ cells per well were allowed to adhere before being treated with gradient concentrations of LNT for 24 h at 37°C, after which they were fixed with 4% paraformaldehyde solution at room temperature for 15 min. Cells were then washed three times with PBS, penetrated with 0.5% Triton X-100 for 20 min, and blocked with donkey serum (cat. no. MB4516; Dalian Meilun Biology Technology Co., Ltd.) for 30 min at room temperature (25°C). Subsequently, they were incubated with a mixture of different primary antibodies, including mouse EGR1 (1:100; cat. no. H00001958-M03; Novus Biologicals, LLC) and rabbit PTEN (1:100; cat. no. AF6351; Affinity Biosciences), overnight at 4°C. The cells were then washed with PBS and incubated with fluorescently-labeled secondary antibody (1:200) for 1 h at room temperature (25°C) in the dark. After staining nuclei with DAPI (1:5,000; cat. no. C1002; Beyotime Institute of Biotechnology) for 5 min at room temperature, cells were sealed with anti-fluorescence quenching sealing solution (cat. no. P0126; Beyotime Institute of Biotechnology). Finally, images were collected using a laser scanning confocal microscope (Leica TCS SP8; Leica Microsystems GmbH).

Secondary antibodies used in IF staining were as follows: Donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody labeled with Alexa Fluor Plus 555 (cat. no. A32794) and donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody labeled with Alexa Fluor 488 (cat. no. A-21202; both from Invitrogen; Thermo Fisher Scientific, Inc.).

All protocols involving animals in the present study were reviewed and approved (approval no. 2020010) by the Animal Ethics and Welfare Committee of Minnan Normal University (Zhangzhou, China). Mice were obtained from Jiangsu Huachuang Xinuo Pharmaceutical Technology (certificate no. 320928211100002573).

In total, 40 female mice (C57BL/6 background) with gestational ages of 11–12 days were housed in a temperature (25°C) and humidity (50–60%)-controlled room, where they were kept on a 12-h light/dark cycle and received autoclaved water and food ad libitum under specific pathogen-free conditions. After the pregnant mice gave birth, 1-week-old mice were acclimatized and fed for 1 week, after which 2-week-old male offspring (weight, ~15 g) were selected. A total of 40 mg/kg DEN (cat. no. HY-N7434; MedChemExpress) was injected intraperitoneally. Following the establishment of the model, mice were randomly divided into the following four groups (8 mice/group): i) Model group; ii) LNT-low dosage group (0.865 mg/kg); iii) LNT-middle dosage group (1.73 mg/kg); and iv) LNT-high dosage group (3.46 mg/kg). Meanwhile, another 8 mice of the same batch without DEN induction were used as the normal group. Each group of mice was treated via intragastric administration for 8 weeks. The normal group and model group were given an equivalent amount of saline. In the late stage of the experiment, the mice in the control group began to present slow movement and hair loss. At the end of the administration, the mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and were sacrificed by cervical dislocation. When cessation of breath and heartbeat was observed, they were subjected to autopsy for observation.

Mouse liver tissues were fixed in a 10% paraformaldehyde solution at 4°C, embedded in paraffin, and sectioned into 5-µm sections. The sections were deparaffinized with xylene and hydrated with gradient ethanol. H&E staining was then performed according to standard techniques. The sections were incubated with a hematoxylin solution (cat no. ZLI-9610; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) for 2 min at room temperature (25°C) and washed with water for 6 min. Then, the sections were soaked in 70% ethanol solution containing 1% hydrochloric acid for 30 sec at room temperature (25°C) and washed with water for 6 min again. Subsequently, they were stained with eosin (cat no. ZLI-9613; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) for 4 min at room temperature (25°C) and rinsed with water for 6 min. Finally, the stained sections were examined using an Olympus fluorescent inverted microscope (IX71; Olympus Corporation).

Tissue expression levels of EGR1 and PTEN proteins were assessed by IHC. Liver tissue sections were deparaffinized with xylene and hydrated with decreasing concentrations of ethanol, after which they were treated for antigen retrieval with 0.01 mM citric acid buffer in a microwave. Endogenous peroxidase activity was inhibited by endogenous hydrogen peroxide (3% H₂O₂) treatment. Sections were then blocked with goat serum (cat no. MB4508-1; Dalian Meilun Biology Technology Co., Ltd.) for 1 h at room temperature (25°C) and incubated overnight at 4°C with rabbit EGR1 (1:200), rabbit PTEN (1:200) or rabbit Ki-67 (1:200; cat. no. ab15580; Abcam). The sections were then treated by IHC kits (cat. no. PV-9001/PV-9002; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) following the manufacturer's protocol. Sections were stained with DAB and counterstained with hematoxylin. Finally, sections were dehydrated in increasing concentrations of ethanol and xylene before being observed and imaged using an Olympus IX71 inverted microscope.

All statistical analyses were performed using GraphPad Prism 8 (Dotmatics). All experiments were repeated independently in triplicate. Data are expressed as the mean ± standard error of the mean (SEM) and analyzed by the independent sample t-test (between two groups) or one-way ANOVA variance analysis with Tukey's multiple comparisons (among-group comparisons), respectively. P<0.05 was considered to indicate a statistically significant difference.

CCK-8 method was used to explore the effect of LNT on the proliferation of Hepa1-6 cells. As revealed in Fig. 1A, the effect of LNT on Hepa1-6 cell viability rate increased with the increasing concentration of LNT. The lower concentration of LNT had no apparent toxicity to cells. However, medium and high concentrations of LNT (500~1,000 µg/ml) had a specific inhibitory effect on Hepa1-6 cell viability rate. The Annexin V-FITC staining method was used to detect the apoptosis of Hepa1-6 cells in situ. Early-stage apoptotic cells were stained with green fluorescence (FITC) only, while late-stage apoptotic cells or necrotic cells were double-stained with green and red fluorescence (PI). However, healthy cells were not fluorescently stained. As demonstrated in Fig. 1B, after the Hepa1-6 cells were treated with increasing concentrations of LNT, the green fluorescence became correspondingly more intense. When the LNT concentration reached 1,000 µg/ml, the degree of early-stage apoptosis appeared the most severe. In addition, flow cytometric analysis demonstrated that the total apoptotic rate of Hepa1-6 cells increased gradually with the increase of LNT concentration, and the LNT treatment at 1,000 µg/ml was more significant. (Fig. 1C). Bcl-2 and Bax, two markers in the Bcl-2 gene family, are closely associated with apoptosis (19). Bcl-2 is considered a representative anti-apoptotic marker (20), whilst Bax is a pro-apoptotic marker in the Bcl-2 family (21,22). After treatment with LNT for 24 h, total protein was extracted from each group for detection using WB. With increasing LNT concentrations, expression of the anti-apoptotic protein Bcl-2 was decreased, whilst expression of the pro-apoptotic protein Bax was increased, resulting in a significant reduction in the Bcl-2/Bax ratio (Fig. 1D). This suggested that LNT could induce apoptosis in Hepa1-6 cells.

A previous study has shown that inhibition of AKT phosphorylation in Hepa1-6 cells and mouse HCC models can cause changes in Bax expression, leading to apoptosis (23). Furthermore, increased expression of PTEN can inhibit AKT phosphorylation, thereby affecting downstream signaling pathways through apoptosis or cell cycle arrest, exerting a tumor suppressor function (24–28). EGR1 is considered a transcription factor of PTEN (29,30). Therefore, the present study next assessed whether LNT treatment can cause changes in the expression of EGR1, PTEN, and Akt phosphorylation in Hepa1-6 cells. From the WB data, it was found that the protein expression levels of EGR1 and PTEN were both significantly increased in Hepa1-6 cells with increasing LNT concentrations. By contrast, the level of Akt phosphorylation demonstrated the opposite trend, particularly p-Akt/Akt ratio decreased significantly (Fig. 2A).

Based on the upregulation of EGR1 expression after LNT treatment in Hepa1-6 cells, cell localization of EGR1 was, therefore, further explored under the same treatment conditions. By co-staining for EGR1 and PTEN with IF, the expression levels and cellular localization patterns of EGR1 and PTEN in Hepa1-6 cells treated with a gradient of LNT concentrations were assessed. These results showed that, in the control group, the weak fluorescence intensity of EGR1 (green) and PTEN (red) was indicative of a low expression level for EGR1 and PTEN. In the LNT-treated group, the fluorescence intensity of EGR1 (green) and PTEN (red) gradually increased, that is, the expression levels of EGR1 and PTEN gradually increased with the increase of LNT concentration, and the green fluorescence emitted by EGR1 was significantly enhanced in the nucleus after administration (Fig. 2B). A subsequent nuclear-cytoplasmic separation experiment revealed that with the increase of LNT concentration, EGR1 expression gradually increased in the nucleus and decreased in the cytoplasm, and the difference was significant (Fig. 2C). These results suggested that LNT could regulate both the expression and nuclear translocation of EGR1 in Hepa1-6 cells. After EGR1 enters the nucleus, it may function as a transcription factor to promote PTEN expression.

To further explore whether EGR1 is an upstream signaling molecule of PTEN and p-Akt, an EGR1 overexpression system was constructed. EGR1, PTEN, p-Akt and Akt expression were all detected by WB. EGR1 overexpression was found to lead to significantly increased PTEN expression and p-Akt/Akt ratio decreased (Fig. 2D). These results suggested that EGR1 was the upstream signal of PTEN and Akt.

Taken together, it was identified that LNT treatment could restore EGR1 expression in mouse HCC cells and allow EGR1 to serve its normal function as a transcription factor, in turn promoting PTEN expression. This then inhibits the AKT signaling pathway, which alters the expression of downstream proteins Bcl-2 and Bax to ultimately trigger the apoptosis of mouse HCC cells.

A primary liver cancer model was established using C57BL/6 mice induced by DEN, after which normal, model and three LNT administration groups were defined. Hyperplastic nodules could be observed on the liver surfaces of mice in the model group compared with those in the normal group. After treatment with LNT, the number of hyperplastic nodules on the liver was significantly reduced in the LNT-treated group compared with that in the model group (Fig. 3A and B). In addition, the tumor volume was significantly decreased (Fig. 3C). H&E staining was used to further observe the pathological situation of tumor tissue. The results revealed that in the model group, the tissue structure of the liver tumor changed significantly and fatty degeneration appeared in certain areas. The morphology and structure of the LNT-treated group were improved compared with the model group. The tumor area was relatively small, and the liver tissue structure in the high-dose group was close to the normal group (Fig. 3D).

In addition, WB and IHC staining were further applied to detect the difference of related protein expression. Changes in the expression of the relevant proteins between the normal and model groups are shown in Fig. 4A. Specifically, compared with the normal group, the model group exhibited significantly reduced expression levels of EGR1 and PTEN, whilst p-Akt/Akt ratio increased, indicating that Akt was activated significantly. The expression levels of proliferation-associated protein PCNA and the anti-apoptotic protein Bcl-2 were significantly increased, whilst those of the pro-apoptotic protein Bax were significantly decreased. The ratio of Bcl-2/Bax, an indicator of cell apoptosis, was significantly increased in the model group. These results suggested that compared with those in the normal liver tissue, there was no significant apoptosis in the liver cancer tissue of the model group, where the expression levels of EGR1 and PTEN were inhibited.

Using WB, it was also identified that compared with the model group the expression levels of EGR1 and PTEN were significantly increased in the LNT-treated group (Fig. 4B). In addition, the p-Akt/Akt ratio was significantly decreased, suggesting that AKT activity was inhibited. The expression levels of Bcl-2 and PCNA expression were significantly decreased, and Bax expression was increased, whilst Bcl-2/Bax ratio decreased. IHC analysis revealed that the protein Ki-67, which is associated with cancer cell proliferation, was markedly reduced with increasing LNT dosages in the LNT administration groups, compared with that in the model group. By contrast, the expression levels of EGR1 and PTEN were markedly increased, with EGR1 mainly distributing to the nucleus (Fig. 4C).

The results of these in vivo experiments are consistent with those found in the in vitro experiments. Therefore, it was considered that LNT could regulate EGR1 expression in mouse HCC cells, supporting its role as a transcription factor that can promote the expression of PTEN. PTEN then inhibits the PI3K/AKT signaling pathway and alters the expression of related apoptotic proteins Bcl-2 and Bax, ultimately inducing apoptosis and inhibiting the development of HCC.