HCC patients and specimens

Human HCC tissues and adjacent non-tumor tissues were provided by Peking University Cancer Hospital (PUCH). All patients involved in this study had signed informed consent forms. The relevant clinical and histopathological data provided to researchers were anonymized. This study was approved by the Ethics Committee of PUCH.

Cell culture

Human HCC cell lines (Huh7 and MHCC-97 H) were obtained from the PUCH. HEK293T cell line was purchased from American Type Culture Collection (ATCC) and maintained by the Peking University Center of Human Disease Genomics. All of these cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, USA) supplemented with 10% Fetal Bovine Serum (FBS; PAN Biotech, Germany) and 1% penicillin-streptomycin (Hyclone, USA). The cells were incubated at 37℃ in a humidified chamber containing 5% CO2.

Cell transfection

Small interfering RNA (siRNA) sequences were directly synthesized (GenePharma, Shanghai, China). Guide RNA (gRNA) sequences were synthesized by Sangon Biotech (Shanghai, China). Short hairpin RNA (shRNA) was purchased from Tsingke Biotech (Beijing, China). His/myc-tagged Vκ4-1/Jκ3 or Vκ1-5/Jκ3 and Flag-tagged ETFA or ETFB were cloned into pcDNA3.1 vector. The siRNA, shRNA and gRNA were transfected into cell lines using Lipofectamine 3000 (Invitrogen, USA), the plasmid DNA were transfected into cell lines using Polyethylenimine (Polysciences, USA) following the manufacturer’s instructions. After transfection, cells were incubated at 37 °C for 6 h before replaced by complete medium and cultured for an additional 48 or 60 h before further assays. The knockdown efficiency was verified by western blot analysis. The sequences of siRNA, shRNA and gRNA are listed in Table S1.

RNA extraction and PCR assay

Total RNA from HCC tissues and cells was extracted using TRIzol reagent (Invitrogen, USA), and cDNA was synthesized from 2 µg of total RNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. To determine the Igκ rearrangement pattern in HCC cells, semi-nested touchdown PCR was performed to amplify IGKV gene transcript, with annealing temperature from 60 °C to 48 °C. The cDNA library of peripheral blood mononuclear cells (PBMCs) was used as a positive control, and reaction systems without templates as a negative control. To determine the expression of FAO-related genes after silencing Igκ in HCC cells, quantitative real-time PCR analysis was performed with Hieff® qPCR SYBR® Green Master Mix (YESEN, Shanghai, China). The expression of the target genes was normalized to GAPDH and determined by the 2−∆∆Ct method. The primers used are shown in Table S2.

Cell proliferation assays

Cell proliferation and viability were assessed using Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Japan) based on the manufacturer’s protocol. Transfected cells were seeded in 96-well plates at a density of 2000 cells per well with 100 µl of medium per well. At designated time points (0, 24, 48, 72 and 96 h), 10 µl of CCK-8 solution was added to each well, and the plate was incubated at 37 ℃ for 2 h. The absorbance at 450 nm was then measured using a microplate reader and recorded.

For colony formation assays, transfected cells were seeded in 12-well plates at 400 cells per well and incubated for 7–10 days. When visible clones appeared, 1% crystal violet was added to the stain at room temperature for 20 min, and the number of clones were counted.

Cell migration assays

For wound-healing assays, transfected cells were seeded in 6-well plates until confluence, and a straight line was scraped using a 200 µl pipette tip. The plate was imaged at the same position and at indicated time points under a light microscope. The relative cell migration rate was analyzed using ImageJ software.

For transwell assays, 250 µl of serum-free medium containing 1.5 × 105 transfected cells were seeded in the upper chambers with 8 μm pore (Corning, USA), and 750 µl complete medium containing 10% FBS was added to the bottom of 24-well plates as a chemoattractant. The cells were maintained at 37℃ for 48 h, and the upper chambers were then removed. The migrated cells on the lower surface of upper chambers were stained with crystal violet and quantified using ImageJ software.

Cell apoptosis detection

Transfected cells were harvested and washed in ice-cold PBS. Suspended cells were then detected in binding buffer with Annexin V-FITC for 15 min and subsequently stained with 7-AAD for 5 min in the dark at room temperature. Cells were detected with a BD FACS Verse instrument (BD Biosciences, USA), and the results were analyzed by FlowJo-V10 software.

Animal studies

The Igκfl/fl mice were mated with Albumin-cre transgenic mice to generate Alb-cre−: Igκfl/fl (wild-type, WT) mice and Alb-cre+: Igκfl/fl (knockout, KO) mice. All experimental procedures were approved by Peking University Laboratory Animal Research Committee and adhered to the Institutional Animal Care and Use Committee of China.

For the HCC mouse model induced by DEN and CCl4, 2-week-old male mice were intraperitoneally injected with 25 mg/kg DEN. At 4 weeks of age, the mice were injected intraperitoneally with 20% CCl4 (5 mg/kg) in corn oil twice a week until 26 weeks. The mice were sacrificed at indicated time point, and the sera were collected for detection. The morphology of mice liver tissue was observed using immunohistochemistry (IHC), hematoxylin and eosin (H&E) and Masson staining.

For the xenograft tumor formation in vivo, the 5 × 106 Huh7 cells transfected with Igκ shRNA or control shRNA in 150 µl of PBS containing 50 µl of Matrigel (Corning, USA) were injected subcutaneously into the flanks of BALB/c nude mice. Tumor growth was monitored every 8 days for the first 38 days and every 4 days thereafter, and the tumor volume was calculated as (width2 × length)/2.

Serum biochemistry

Blood was collected from HCC mouse model, and the serum level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was measured using standard enzymatic procedures according to the manufacturer’s instructions (C009-2-1, C010-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Histology and immunohistochemistry (IHC)

The liver pathology of HCC mouse model was examined. Liver tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. Tissue sections were stained with H&E and Masson (D026-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions to observe the level of tissue damage using light microscopy.

Liver tissues of human and mouse were subjected to immunohistochemistry. Tissue sections were deparaffinized in xylene, dehydrated in an ethanol gradient, and subjected to antigen retrieval in 10 mM citrate buffer (pH 6.0) in a heater. To inactivate endogenous peroxidase activity, the sections were treated using a 3% peroxidase solution for 10 min and then blocked with goat serum at 37 °C for 1 h. The sections were incubated with indicated antibodies at 4 °C overnight, followed by the incubation with HRP-conjugated anti-mouse/rabbit IgG at RT for 30 min. The immunoreactivity was visualized using an enhanced diaminobenzidine kit (Dako, Denmark) and nuclear staining with hematoxylin. The expression level was quantified using a four-tier intensity score (0, none; 1, weak; 2, moderate; 3, strong) and the percentage (0-100%) of positive cells. The final staining score was obtained by multiplying the scores for the intensity and percentage of positive cells (range, 0-300). Scores > 100 were defined as high, while scores ≤ 100 were defined as low.

Western blot analysis

Total proteins were extracted from HCC tissues or cell lines using RIPA buffer (Beyotime, Shanghai, China) with a complete protease inhibitor cocktail and phosphatase inhibitors (Roche, Basel, Switzerland). Aliquots of protein extracts were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk for 1 h at room temperature and incubated with primary antibodies overnight at 4 ℃. After washing with TBST, membranes were incubated with peroxidase-coupled secondary antibodies for 1 h at room temperature. Protein bands were visualized using an ECL kit (Thermo Fisher Scientific, USA) and iBright™ CL750 imager. The antibodies used in this assay are listed in Table S3.

Co-immunoprecipitation (Co‑IP) assay and mass spectrometry

Huh7 cell lysates were prepared in lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, pH 7.5), and incubated with anti-Vκ4-1/Jκ3 antibody or isotype control overnight at 4 ℃ with rotation, followed by immunoprecipitation with protein G beads at 4 ℃ for 4 h. After extensive washing to remove the non-specific binding proteins, precipitates were examined by western blot analysis using the indicated antibodies. Mass spectrometry was performed at the Institute of Biotechnology of Peking University Health Science Center (Beijing, China).

For Co-IP assays, protein lysates were prepared from HEK 293T cells in a buffer containing 50 mM Tris, 150 mM NaCl, 1% NP-40 (pH 7.5), and a mixture of protease and phosphatase inhibitors. The lysates were then incubated with anti-His-tag mAb-Magnetic Beads (MBL, Japan) or anti-Flag-tag mAb-Magnetic Beads (Abmart, Shanghai, China) at 4 ℃ for 2 h. After washing with wash buffer (50 mM Tris, 150 mM NaCl, 0.1% NP-40), the precipitates were analyzed by western blot analysis.

Immunofluorescence (IF) staining

Slides were seeded in the bottom of 24-well plate, and when cell confluence reached 70–80%, the medium was discarded and the cells were gently washed with PBS three times. To determine the cell localization of ETFA, Mito-Tracker Red CMXPos (Beyotime, Shanghai, China) was used as a positive control for mitochondrial staining. The pre-heated Mito-Tracker Red CMXRos solution (200 nM) was added to the cells and incubated at 37℃ for 30 min. The cells were then fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After blocking with 10% goat serum, the cells were incubated with primary antibody overnight at 4 ℃. After washing with PBS three times, the fluorescent secondary antibodies were added, and the samples were incubated for 1 h at room temperature in the dark. Finally, the cells were mounted with a DAPI-containing mounting medium that resists fluorescence quenching (Vector Labs, USA), and images were observed and collected using a confocal microscope with an Olympus FV3000 imager.

RNA sequencing

Total RNA from control or Igκ knockdown Huh7 cells was isolated for RNA sequencing (RNA-seq), which was performed by Novogene Company (Beijing, China). Differentially expressed genes were analyzed using the cloud platform of Novogene. A P value < 0.05 and log2 fold change > 1 were considered to meet the screening criterion, and the differential genes were enriched by KEGG analysis.

Label-free proteomics analysis

Total protein was extracted from transfected Huh7 cells and subjected to LC-MS/MS analysis at the Institute of Biotechnology of Peking University Health Science Center (Beijing, China). Label-free quantification (LFQ) was used to compare the abundance of proteins between the Igκ-knockdown and control groups, with GAPDH used to normalize each sample’s LFQ. Significantly altered proteins (p value < 0.05 and log2 fold change > 1.5) were subjected to GO-BP (biological process) analysis using the website of https://metascape.org/gp/index.html#/main/step1. Differential proteins are shown by a heat map using R software.

Untargeted metabolomic analysis

Transfected Huh7 cells were collected and stored at -80 ℃ before UPLC-Q-TOF/MS analysis at Igenecode Technology Company (Beijing, China). For LC-MS analysis, samples were treated with 1 mL of cold methanol/acetonitrile/H2O (2:2:1, v/v/v) and centrifuged at 13,000 rpm and 4 ℃ for 15 min to obtain supernatants. The supernatants were mixed with 100 mL acetonitrile/ H2O (1:1, v/v). Analyses were performed using a UHPLC system (1290 Infinity LC, Agilent Technologies, USA) coupled to a quadrupole time-of-flight instrument (AB Sciex TripleTOF 6600). The stability and repeatability of the instrument analysis were monitored using quality control (QC) samples.

Metabolite measurement

Transfected Huh7 cells seeded in 10 cm dishes were grown to 80% confluence and used for the measurements of various metabolites. Cells were treated with n-Heptane: isopropanol (1:1) for triglyceride measurement, n-Heptane: absolute methanol: chloroform (24:1:25) for free fatty acid measurement or extracting solution for ATP measurement, and then sonicated. After centrifugation, the supernatant was used to determine triglyceride level using a Triglyceride Levels Assay Kit (BC0652), Free Fatty Acid Levels Assay Kit (BC0595) or an ATP Levels Assay Kit (BC0305) purchased from Beijing Solarbio Science & Technology Co. LTD (Beijing, China) according to manufacturer’s instructions.

Oil Red O staining assay

Oil Red O (ORO) staining was performed with an Oil Red O Kit (G1262, Beijing Solarbio Science & Technology Co. LTD, China). Cells were washed with PBS twice and fixed with the fixative buffer for 30 min. Then cells were washed with distilled water twice and then incubated in 60% isopropanol for 1 min. The newly prepared Oil Red O staining solution was added and soaked for 20 min. Mayer hematoxylin staining solution was added for 2 min. After washing, the cells were observed and photographed under a light microscope (BZ-X800, Japan).

BODIPY™ 493/503 staining assay

Cells were fixed with 4% paraformaldehyde, washed with PBS, and stained with 0.2 ng/µl BODIPY™ 493/503 (Thermo Fisher Scientific, USA) at room temperature for 15 min. The cells were then mounted with a DAPI-containing mounting medium to visualize the nuclei, and the images were observed and collected under a fluorescence microscope (Olympis FV3000, Japan).

Statistical analysis

The data were statistically analyzed and graphed with Prism software version 8 (GraphPad, CA, USA). The differences between two groups were determined using two-tailed t tests. One-way ANOVA and two-way ANOVA were used to analysis the differences between univariate and multivariate variables among the three groups of data. Kaplan-Meier and log-rank tests were used to determine survival rates. All statistical tests were two-sided probability tests. Significance was indicated as follows: ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.