Cell lines and human tissue

HCC cells (Huh7, MHCC97L, HepG2, Hep3B, HCCLM3, Focus, and YY8103), immortalized human hepatocyte THLE-3 cells, and HEK293T cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China).

HCC tissues and adjacent non-cancerous samples were obtained from patients who underwent hepatic resection at the Hepatobiliary Center of the First Affiliated Hospital of Nanjing Medical University. All participants in this study provided written informed consent, which was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. The human experiments were conducted in accordance with the ethical norms of the World Medical Association, as stated in the Declaration of Helsinki.

Cell culture and transfection

All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, USA) and 50 U/ml penicillin‒streptomycin (Invitrogen). The cells were cultured in a 5% CO2 incubator at 37 °C.

Plasmid and small interfering RNA (siRNA) transfections were performed using Lipofectamine 3000 (Invitrogen) according to the provided protocol. For lentiviral transfections, polybrene (Invitrogen) was used to enhance transfection efficiency. Puromycin (Invitrogen) was employed to select stable transfected cells. The efficiency of transfection was evaluated through quantitative real-time PCR (qRT-PCR) and western blot analysis. Lentiviruses overexpressing BAIAP2L2, lentiviruses containing BAIAP2L2 small hairpin RNA (shRNA), siRNA targeting NFκB1, an overexpression plasmid for NFκB1, siRNA targeting GABPB1, and an overexpression plasmid for GABPB1 were obtained from GenePharma (GenePharma, Shanghai, China). The shRNA and siRNA sequences are provided in Table S1.

Quantitative real-time PCR (qRT-PCR)

Total RNA from HCC tissues and HCC cell lines was isolated using an RNA extraction kit (Invitrogen). Then, the total RNA was transcribed to cDNA with a Prime Script RT kit (TaKaRa, Dalian, China). qRT-PCR was performed using SYBR Premix ExTaq II (TaKaRa) and the ABI 7900 PCR system (Applied Biosystems, CA, USA). The results were calculated using the 2−ΔΔCT method, and the primer sequences are listed in Table S2.

Tissue microarrays (TMAs)

The HCC tissues and adjacent non-cancerous tissues were obtained from the Hepatobiliary Center of the First Affiliated Hospital of Nanjing Medical University. Human HCC TMAs were created by Servicebio (Wuhan, China).

Immunohistochemical (IHC) staining

The specimen was fixed in formaldehyde, followed by embedding in paraffin and subsequent sectioning. Deparaffinization was performed using xylene and anhydrous ethanol, after which rehydration was carried out through a gradient of ethanol concentrations. Antigen retrieval was achieved by boiling the sample in a sodium citrate solution for 30 min. Blocking was accomplished with goat serum, and the primary antibody was added, with incubation occurring overnight at 4 °C. The sections were then washed with TBST, and after applying the secondary antibody, incubation was conducted at 37 °C for 30 min. Staining was performed using a DAB chromogen (Beyotime), followed by hematoxylin for nuclear visualization.

Cell counting kit-8 (CCK-8) analysis

Transfected cells were seeded in a 96-well plate. CCK-8 solution (RiboBio, Guangzhou, China) was added every 24 h according to the manufacturer’s protocol. The absorbance of each group was subsequently measured at a wavelength of 450 nm. Continuous testing was performed for 5 days.

Wound-healing assay

The wound-healing assay was performed to detect the cell migration capacities. In brief, the transfected cells were plated in 6-well plates, and the cell surface was scratched using a 200 μL plastic pipette tip. Wound closure was monitored at 0 and 48 h.

Transwell assay

In the cell migration experiment, 2 × 104 cells were seeded in the upper chamber of a transwell chamber (Corning, NY, USA) with 200 μL of serum-free DMEM. Then, 600 μL of DMEM containing 10% FBS was added to the lower chamber. For the cell invasion experiment, Matrigel (diluted 1:8; BD Biosciences, Franklin Lakes, USA) was added to the upper chamber, and the remaining steps were identical to those in the migration experiment. Incubation occurred for 48 h. The upper cells were removed, and the remaining cells were fixed and stained for quantitative analysis.

5-Ethynyl-20-deoxyuridine (EdU) assay

Transfected cells were incubated with an EdU solution (RiboBio) in 24-well plates for 2 h. Then, the cells were fixed and the aldehyde groups were neutralized with 2 mg/mL of glycine. Cells were permeabilized with 0.5% Triton X-100 and then stained with Apollo and DAPI.

Sphere-forming assay

A total of 2000 cells were seeded in a 24-well suspension cell culture plate (Corning) containing 2 mL of stem cell culture medium. Stem cell culture medium: DMEM/F12 (Gibco) supplemented with 1X B27 (Sigma-Aldrich, Saint Louis, USA), 20 ng/mL EGF (PeproTech, NJ, USA), 20 ng/mL FGF (PeproTech), and 4 µg/mL heparin. The cells were cultured for 10 days. The formation of pellets by the cells was observed, and the size and number of pellets were calculated.

Construction of hepatocellular carcinoma organoid model

The organoid culture protocol followed a previously established methodology [11]. Patient-derived specimens were minced and subsequently incubated in a digestive solution for 4 h at 37 °C. Then, the cells were combined with Basement Membrane Extract (BME) (R&D Systems, MN, USA) on ice, achieving a concentration of 10,000 cells in 40 µl of BME. Approximately 10 µl droplets of BME were then deposited into each well of a 24-well suspension cell culture plate, after which 400 µl of organoid medium was added to each well. The organoids were maintained in a 37 °C incubator with an atmosphere of 5% CO2, and the culture medium was refreshed biweekly.

Western blot assay

Cells were lysed with prechilled radioimmunoprecipitation (RIPA) buffer, supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). After adding a loading buffer and boiling for 10 min to denature the protein, the proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA, USA). The proteins were incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 2 h at room temperature. The visualization was performed using ECL (Yesen, Shanghai, China) detection. The antibodies are listed in Table S3.

Flow cytometry analysis

In the cell cycle analysis, after fixing the cells, 5 × 105 cells were added to each tube and stained with propidium iodide (PI) (Vazyme, Nanjing, China). Cell cycle distribution was measured using a BD FACS Canto II. In the cell apoptosis analysis, transfected cells were stained with Annexin V-FITC solution (Vazyme) and PI solution, following the manufacturer’s instructions. Apoptosis was detected using the BD FACS Canto II.

Animal models

In the present study, ethical clearance for all animal experimentation was obtained from the Institutional Animal Care and Use Committee (IACUC) at the First Affiliated Hospital of Nanjing Medical University. All animal-related procedures were conducted in accordance with the operational protocols established by the IACUC. BALB/c nude mice (4 weeks old, male) were acquired from Vital River (Beijing, China).

For the subcutaneous tumor model, the BALB/c nude mice were randomly assigned to four groups, each consisting of five mice. The transfected cells were injected into the flanks of nude mice, and the tumor size was recorded every 3 days for 4 weeks. The volumes of the tumors were calculated using the formula: length × width2/2.

For the orthotopic tumor transplantation model, the necessary cells were prepared as a 5 × 107/mL cell suspension. After successful isoflurane anesthesia, the nude mice were disinfected below the xiphoid process along the mid-abdominal line, and an ~0.5 cm incision was made to expose the left lobe of the liver. A total of 20 µl of cell suspension was injected beneath the liver capsule. At week 4, the fluorescence distribution and intensity of the liver in the abdominal region were observed. Subsequently, the nude mice were sacrificed for liver analysis.

The lung metastasis model was established through tail vein injection. There were 15 nude mice in each group, and each mouse was injected with ~1 × 106 post-transfected cells through the tail vein. At week 4, five mice per group were sacrificed. Survival analysis was performed on the remaining mice using a 12-week cutoff.

Luciferase reporter assay

The BAIAP2L2 promoter sequence was obtained from the UCSC database (http://genome.ucsc.edu/) and the BAIAP2L2 promoter regions (−2000/0, −1500/0, −1000/0, −750/0, −500/0 and −250/0) were inserted into the pGL3-Basic vector (Promega, Madison, USA) to construct a luciferase reporter gene. Two single-site mutation (−522 ~ −532) mut and (−984 ~ −994 bp) mut and two-site mutation (−522 ~ −532 mut and −984 ~ −994 bp) sequences of BAIAP2L2 promoter were inserted into the pGL3-Basic vector. The relevant plasmids were transfected into 293T cells using Lipofectamine 3000 (Invitrogen). Dual-luciferase reporter gene assays were conducted using the Dual-Luciferase Reporter Assay System (Promega).

Chromatin immunoprecipitation assays

A chromatin immunoprecipitation (ChIP) Assay Kit (Beyotime, Shanghai, China) was used to perform ChIP assays. Briefly, following cell lysis, the chromatin was fragmented to sizes ranging from 200 to 2000 base pairs using an ultrasonic cytometer, and then immunoprecipitated with anti-NFkB1 and IgG antibodies. The enriched DNA templates were analyzed by PCR using primer pairs specific to the target gene promoter (Supplementary Table 2).

Coimmunoprecipitation (Co-IP) assay

In the conducted Co-IP experiments, Anti-Flag Magnetic Beads (MedChemExpress, NJ, USA) were utilized. Plasmids containing flag-tagged BAIAP2L2, flag-tagged GABPB1, and flag-tagged truncated BAIAP2L2 were transfected separately into HCC cells. The magnetic beads were incubated overnight with the transfected cell lysates. Following the elution of the magnetic beads, protein interactions were analyzed using western blotting.

Ubiquitination detection assay

Transfected cells were treated with 20 μM MG132 (Beyotime) for 6 h. After that, the total protein was collected. The supernatant was harvested and immunoprecipitated using anti-Flag Magnetic Beads. Western blotting was performed using an anti-HA-Ubiquitin antibody to detect the ubiquitination of GABPB1.

Immunofluorescence assay and confocal microscopy

Cells cultured on confocal dishes were fixed and permeabilized for 10 min. The rabbit antibody BAIAP2L2 and mouse antibody GABPB1 were added and incubated overnight. Then, cells were stained with Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG. Nuclei were labeled with DAPI, and cells were visualized using a confocal laser scanning microscope (Olympus).

RNA sequencing

Three pairs of BAIAP2L2-overexpressing Hep3B cells and negative control cells were lysed with TRIzol reagent. Next, RNA sequencing (JI GUANG Gene, Nanjing, China) was performed.

Mitochondrial membrane potential and reactive oxygen species analysis

To assess the alterations in mitochondrial membrane potential, we employed the Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime) following the instructions provided by the manufacturer. Intracellular levels of ROS were evaluated using a Reactive Oxygen Species Assay Kit (Beyotime) according to the manufacturer’s instructions. Images were obtained using a fluorescence microscope.

Establishment of lenvatinib-resistant cell line

Huh7 cells were cultured in a 96-well plate at a density of 2000 cells/well and treated with increasing concentrations of Lenvatinib (MedChemExpress). CCK-8 assay was used to determine the half-maximal inhibitory concentration (IC50) of lenvatinib on Huh7 cells after 48 h. Subsequently, the lenvatinib-resistant Huh7 cells (Huh7 LR) cells were cultured either in the absence of lenvatinib or with 10 μM of lenvatinib for a duration of 1 month. The IC50 of lenvatinib in Huh7 LR cells was measured again to confirm that Huh7 LR cells are long-term resistant to lenvatinib.

Statistical analysis

The statistical analysis was conducted using SPSS version 22.0 (Chicago, IL, USA) and GraphPad Prism version 7.0 software (La Jolla, CA, USA). The differences between the two groups were analyzed using Student’s t-test. The clinical characteristics were assessed using the Chi-square test, while correlation analysis was conducted with the Spearman correlation coefficient. Quantitative data were presented as the mean ± standard deviation. Survival rates were plotted using Kaplan–Meier estimates, and Cox proportional hazards regression analysis was performed to identify independent factors for prognosis. Statistical significance was defined as p<0.05 (*), p<0.01 (**), and p<0.001 (***).