2.1 Reagents

Peptides containing an α-helix corresponding to the TRIB3 117–220 amino acid region and R164A/Q175A (2A) mutant peptides fused with a cell-penetrating peptide (Pep, HLYVSPWGG) at the N terminus were synthesized at Chinapeptides Co., Ltd (Shanghai, China). The sequences of peptides used in this study were as follows:

P-T3H2 (HLYVSPWGGEPEAAVLFRQMATAHCHQ),

P-T3H2-2A (HLYVSPWGGEPEAAVLFAQMATAHCHA).

2.2 Patients and clinical specimens

The human HCC tissues were obtained from the Eastern Hepatobiliary Surgery Hospital and Changzheng Hospital, Naval Medical University (Shanghai, China). All patients were diagnosed with HCC by postoperative pathology. The informed consent was obtained from all patients before treatment. All experiments involving human samples was conducted with the approval of the Medical Research Ethics Committee of Naval Medical University.

2.3 Western blotting analysis

The protein of cells or tissues were extracted with lysis buffer (125 mM Tris–HCl pH 6.8, 25% glycerol, 5% SDS) supplemented with 1/100 protease inhibitor (Beyotime, China). The protein samples were separated using 10% SDS-PAGE gel and transferred onto nitrocellulose membranes (HAHY00010, Millipore). The membranes were blocked in PBST (Phosphate-Buffered Saline with Tween) containing 5% skim milk for 1 h after transfer and incubated with the indicated primary antibody(HNF4α antibody, Abcam, ab181604; HNF4α antibody, Sigma-Aldrich, HPA004712; β-Actin antibody, abclonal, AC038; TRIB3 antibody, Abcam, ab50516; TRIB3 antibody, Millipore, ST1032) overnight at 4 °C. After washing three times by PBST, the membranes were incubated with the secondary antibody (donkey-anti-mouse or donkey-anti-rabbit, IRDye 700 or IRDye 800, respectively). Finally, the protein bands were visualized using an Odyssey infrared imaging system (LI-COR) at 700 nm or 800 nm.

2.4 Immunohistochemistry

Formaldehyde-fixed, paraffin-embedded sections of tissues were used for histopathological examination. The tumor sections were successively deparaffinized in xylene and rehydrated in varying alcohols, and blocked for endogenous peroxidase with 3% H2O2 before antigen retrieval. Then the sections were exposed to primary antibodies overnight at 4 °C. After washing with PBST three times, the sections were exposed for an hour to a secondary antibody at room temperature. The antibodies used for IHC staining were including mouse anti HNF4α (Perseus Proteomics, PP-K9218), TRIB3 (abcam, ab50516) and mouse anti-Ki67 (BD Pharmingen, AB393778).

2.5 Cell culture and treatment

The human HCC cell lines Huh7, Hep3B and MHCC-L were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Hep3B cells were cultured in Eagle’s minimum essential medium (MEM) with 10% FBS and nonessential amino acid (NEAA). The cells were cultured at 37 °C in a humidified incubator containing 5% CO2. After cells attachment, the cells were treated with indicated concentrations (20 μM) of P-T3H2-2A or P-T3H2. Peptides were supplemented every 48 h.

2.6 Cell proliferation assay

Huh7 and Hep3B cells were seeded in 96-well plates at a density of 3000 cells/well. The cells were treated with PBS, 20 μM of P-T3H2-2A or P-T3H2. The medium was changed every 2 days and supplemented with corresponding concentration of peptides. According to the manufacturer’s instructions, Cell Counting Kit-8 (CCK-8; Dojindo Laboratories) was added into the wells at the indicated time points. After an incubation of 1 h at 37 °C, absorbance was measured at 450 nm using a microplate reader (BioTek Instruments).

2.7 Cell migration and invasion assay

Cell migration and invasion assays were conducted by Transwell chambers (BD Bioscience), without or with Matrigel, according to the manufacturer’s instructions. In briefly, 3 × 104 Huh7 and Hep3B cells were seeded in the upper chamber which contained 20 μM of P-T3H2-2A or P-T3H2. In the lower chamber, medium was supplemented with 10% FBS and the same concentration of indicated peptide. After incubation for 72 h, the cells on the lower surface of the membrane were fixed and stained with 0.1% crystal violet. Image analysis was performed using Image-Pro Plus 6.0 software (Media Cybernetics) to measure the area of staining.

2.8 Real-time PCR

Total RNA was isolated by TRIzol (Takara) in the standard protocol. The cDNA synthesis of cell was performed using the RT Master Mix (Takara). The cDNA synthesis of tissues was using M-MLV Reverse Transcriptase (Promega). Transcript levels were detected using SYBR Green-based real-time PCR performed by the ABI StepOne Real-time PCR Detection System (Life Technologies). Each condition was tested in at least three independent experiments. The primer sequences are provided in TableS1.

2.9 Periodic acid-Schiff (PAS) staining

Huh7 and Hep3B cells were plated in 24-well plates at 2 × 104 cells/well. After treating with 20 μM of P-T3H2-2A or P-T3H2 for 3 days, the cells were stained with a PAS Reaction Kit (Beyotime, China) following the manufacturer’s protocol. Briefly, the cells were fixed in 75% ethanol for 10 min and oxidized for 10 min with periodic acid. Then the cells were submerged in Schiff’s reagent for 70 min after washing with PBS. Last, the cells were added 100 μl of hematoxylin staining solution and stained for 20 s. Light microscope was used to visualize the glycogen storage. Image analysis was conducted by Image-Pro Plus 6.0 software (Media Cybernetics) to measure the area of staining.

2.10 Senescence associated β-galactosidase (SA-β-gal) activity assay

Huh7 and Hep3B cells were plated into 6-well plates at 3 × 105 cells/well and treated with 20 μM of P-T3H2-2A or P-T3H2 for 4 days. To evaluate cellular senescence, we employed a Senescence β-Galactosidase Staining Kit (Beyotime, China). The cells were first fixed with 4% paraformaldehyde for 15 min and incubated with fresh senescence-associated β-galactosidase staining solution at 37 °C overnight. The senescent cells were then visualized and captured under the microscope. Quantitative analysis of the stained area was conducted using Image-Pro Plus 6.0 (Media Cybernetics).

2.11 Acetylated low-density lipoprotein (ac-LDL) uptake assay

2 × 104 cells/well Huh7 and Hep3B cells were inoculated into 24-well plates. Subsequently, the cells were treated with 20 μM of P-T3H2-2A or P-T3H2 for 3 days. Then medium containing Dil-Ac-LDL (Invitrogen) at a ratio of 1:100 was added into each well for 3 h at 37 °C. After staining the nuclei with DAPI (Sigma, D9542), each well was photographed by fluorescence microscopy.

2.12 Luciferase reporter gene assay

8 × 104 cells/well Huh7 and Hep3B cells were transfected with a reporter plasmid containing 9 copies of the HNF4α binding site from the NINJ1 promoter (5ʹ-CAGGAAAACCTAAGGTCAGGGAG-3ʹ), pGL3-NINJ1-9p, which was constructed by our lab [12], and treated with PBS, 20 μM of P-T3H2-2A or P-T3H2 for 48 h in 96-well plates. To normalize the data, the pRL-SV40 (Promega, E2261) vector was transfected into all the treated cells. Luciferase reporter gene assays were performed using the Dual-Luciferase Reporter Assay System (Promega, E2920) according to the manufacturer’s instructions.

2.13 Animals

Male BALB/c nude mice and C57B/6J mice were purchased from Shanghai BK/KY Biotechnology company. All animal experiments in this study were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Medical Research Ethics Committee of Naval Medical University.

2.14 Human HCC xenograft model

6 weeks old male BALB/c nude mice were purchased from Shanghai BK/KY Biotechnology company and used for HCC xenograft models. All the mice were provided hygienic food and water. All mice were subcutaneously injected on the right flank with 1 × 106 Huh7 cells suspended in 100 μl DMEM. Upon tumors reaching an average volume of about 150 mm3, the mice were randomly assigned to three groups (seven animals per group). Treatments included intratumoral injections with PBS, 5 mg/kg of P-T3H2-2A or P-T3H2, administered twice a week. The volume of tumor was measured every day post-injection. The volume was calculated by the following equation: volume = length × (width)2 × 1/2. Following 3 weeks of treatment, euthanasia was performed on mice using an overdose of pentobarbital (250 mg/kg via intraperitoneal injection). Once the death of the mice was confirmed due to respiratory and cardiac arrest, tumors were excised and weighed.

For the orthotopic HCC model, 1 × 106 Huh7 cells labeled with luciferase were injected subcutaneously into the right flank of nude mice to generate tumor xenografts. Then the tumor nodules from the subcutaneous xenograft model were cut into 1 mm3 pieces and implanted into the left lobe of the livers of nude mice (male, 6 weeks old) to mimic primary HCC. Mice were randomly divided into two groups (six animals per group) according to radiance counts of bioluminescence after implantation. The tumor bearing mice were administrated with 5 mg/kg of P-T3H2-2A or P-T3H2 twice a week via tail vein. To monitor tumor size, mice were intraperitoneally injected with d-luciferin potassium salt with the dosage set at 150 mg/kg. Caliper IVIS Lumina bioluminescence imaging system (PerkinElmer, Waltham, MA, USA) was used to image the mice after a 3-min interval injection. Bioluminescence imaging was repeated every 3 days. The mice were euthanized after 3 weeks of treatment. In our animal studies, we confirm that the maximal tumor size/burden did not exceed the standard (20 mm diameter) set by the Medical Research Ethics Committee of Naval Medical University.

2.15 Primary HCC model

To generate diethylnitrosamine (DEN)-induced mouse primary HCC model, 2 weeks old male C57B/6 J mice were treated with DEN (25 mg/kg), followed by intraperitoneal injection with 10% CCl4 (dissolved in oil, 0.5 ml/kg) weekly from the age of 6 weeks. The mouse was sacrificed at the age of 16 weeks, 18 weeks and 20 weeks, and tumor tissues were harvested respectively.

To establish the spontaneous mouse HCC model, we performed hydrodynamic injection of plasmid DNAs into 6 weeks old male C57B/6J mice via tail vein following previous study [23, 24]. Each mouse was received 20 µg pT3-EF1α-c-MET (human), 20 µg pT3-EF1α-ΔN90-β-catenin (human) and 1.6 µg pCMV-SB, encoding the sleeping beauty transposase and transposon. Mice were treated with 5 mg/kg of P-T3H2-2A or P-T3H2 twice a week via tail vein and sacrificed at 8 weeks after plasmid injection. The liver weight, body weight and liver tumor numbers were measured.

2.16 RNA-sequencing

Total RNA from Huh7 cells treated with 20 μM of P-T3H2-2A or P-T3H2 was collected with TRIZOL reagent. Subsequently, RNA sequencing was executed on the Illumina Novaseq6000 platform using RNA libraries that had been prepared for sequencing in accordance with normal Illumina techniques. The amount of reads that were mapped to each gene was detected by the HTSeq v0.6.0 program. After performing significance analysis and FDR analysis under the following conditions: (i) Fold Change > 2 and (ii) p-value 0.05, DESeq2 method was used to select the differentially expressed genes. Gene Ontology (GO) enrichment analysis was then utilized to classify the biological functions of distinct genes in the profiles of differentially expressed genes. Fisher’s exact test was employed to single out significant GO terms or pathways within an FDR-defined threshold of significance. KEGG pathway analysis was conducted to identify differentially expressed genes. Finally, Gene Set Enrichment Analysis (GSEA) was utilized to identify pathways enriched in HNF4α target gene sets in the sequencing data.

2.17 Statistical analysis

All data were analyzed with Prism 8 (GraphPad Software, La Jolla, CA). Comparisons of data between two groups were performed by Student’s t-tests. The data for animals were presented as the mean ± SEM. Statistical tests were two-tailed and a p < 0.05 was considered statistically significant.