UBE2Q2 promotes tumor progression and glycolysis of hepatocellular carcinoma through NF-κB/HIF1α signal pathway

2.1 Clinical HCC patients’ samples and information

Tumor and adjacent normal liver tissues of 80 HCC patients (68 male and 12 female patients) on whom the liver resections were performed during 2018 to 2020 in the Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University were collected. None of the patients received any pre-operative therapy like radiofrequency ablation, anhydrous alcohol injection, TACE or immunotherapy. The histopathological examination of samples was confirmed by two experienced pathologists. Related clinical data of 80 patients were recorded for statistical analysis.

2.2 Cell culture

The cell lines Huh-7, HCC-LM3, Hep3B, HepG2, HepG2.2.15, and SK-Hep-1 were obtained from the Shanghai Cell Bank of the Chinese Academy of Medical Sciences. These were cultured under standard conditions: Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, maintaining a temperature of 37 °C and a 5% CO2 atmosphere. Cell line authenticity was verified through STR profiling and all were certified mycoplasma-free.

For specific experiments, Huh-7 and LM3 cell lines underwent treatment with distinct agents: 100 µM cobalt chloride for 12 h; 10 µM chlorhexidine, corynoxeine, or LY294002, each for a period of 24 h; 20 µM rapamycin for 24 h; 20 µM (-)-DHMEQ for 24 h and 2 mM 2-DG for 12 h. All aforementioned compounds were sourced from Sigma Aldrich (Germany) and MCE (USA).

2.3 RNA extraction, reverse transcription, and quantitative real-time PCR (qRT-PCR)

RNA from both tissue specimens and cultured cells was isolated using TRIzol Reagent (Invitrogen, USA). The quality and concentration of RNA were assessed with a NanoDrop ND2000 spectrophotometer (Thermo Scientific, Massachusetts, USA). Reverse transcription was conducted using the HiScript III 1st Strand cDNA Synthesis Kit for mRNA and the Mir-X miRNA First-Strand Synthesis Kit (Takara, USA), following the provided protocols. Quantitative real-time PCR was performed with a SYBR Green PCR kit (Vazyme, China) on a CFX96TM Real-time PCR Detection System (Bio-Rad, California, USA). Relative RNA expression levels were quantified using the 2^(-ΔΔCt) method, with the threshold cycle (Ct) being determined. Reproducibility was ensured by conducting all procedures in triplicate. Primer sequences used are detailed in Table S1 of the supplementary materials.

2.4 RNA interference and cell transfection

UBE2Q2 expression was targeted for knockdown using small interfering RNAs (siRNAs). Three distinct siRNAs aimed at UBE2Q2 (referred to as siRNA #1, siRNA #2, and siRNA #3) were custom-synthesized by Genecreate (Wuhan, China). Among them, siRNA #2 was subsequently labeled as siUBE2Q2 in Figs. 4 and 5 and Fig. S4. The details of these siRNA and shRNA sequences are provided in Table S2 of the supplementary materials. For the siRNA transfection, the Genmute transfection reagent (Signagen, Maryland, USA) was used, following the manufacturer’s protocol.

2.5 Construction of plasmid, lentivirus package and cell infection

The UBE2Q2 gene was synthesized and cloned into the pEZ-Lv105 vector to create an overexpression construct, hereafter referred to as UBE2Q2 plasmid. The empty pEZ-Lv105 vector was utilized as a control, designated as Vector. Both plasmids were products of GeneCopoeia (Rockville, USA). Additional plasmids, including pcDNA-HA-UBE2Q2, pcDNA-HIF1α, pcDNA-His-TRAF2, and pcDNA-Flag-cIAP1, were synthesized by Genecreate (Wuhan, China). Plasmids pCDEF-HA-Ub(K11), pCDEF-HA-Ub(K48), and pCDEF-HA-Ub(K63) were procured from Ke Lei Biological Technology (Shanghai, China). Transfections were carried out using Lipofectamine 3000 (Thermo Fisher Scientific, CA, USA) according to the manufacturer’s recommendations.

Stable UBE2Q2 knockdown and overexpression lines were established using lentiviral systems (Lv-shUBE2Q2 and UBE2Q2 respectively), also provided by Genecreate (Wuhan, China), with a virus titer of 6 × 108 TU/mL. For infection, a multiplicity of infection (MOI) of 10 was applied to Huh-7 cells, with 100 µL of viral suspension coupled with 100 µL of Hitrans P virus infection reagent (GeneChem, Shanghai, China) per 1 × 106 cells. After 24 h, the medium was replenished. Cells were collected 4 days post-infection and selected with puromycin. The knockdown efficiency was evaluated using qRT-PCR and Western blot analyses.

2.6 Cell viability assays

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Biosharp, China) assay. Briefly, cells were seeded at a density of 5,000 per well in 100 µL of culture medium in a 96-well plate. Afterward, 10 µL of CCK-8 solution was added to each well. The plate was incubated at 37 °C for 1 h before measuring the absorbance at 450 nm using a microplate reader.

2.7 Colony formation assays

A colony formation assay was performed by plating 500 to 1,000 cells in a six-well plate and culturing for 14 days. The colonies were then fixed with 4% paraformaldehyde and stained for 1 h with 0.1% crystal violet (Sigma Aldrich, Germany). Post-staining, colonies were counted and imaged using a stereomicroscope.

2.8 Wound healing assay

Cell transfection and culture were performed in a six-well plate. Upon reaching confluence, a 100 µL pipette tip was used to create a straight scratch through the cell monolayer. Subsequently, cells were incubated in serum-free DMEM. Wound closure was documented at 0 h, 24 h and 48 h post-scratch, and analyzed using Image J software.

2.9 Cell invasion and migration assays

Migration and invasion assays were conducted using a 24-well plate with a Transwell insert (8 μm pore size, BD Biosciences, USA). Medium with 10% FBS (600 µL) was added to the lower chamber. For invasion assays, the upper insert was precoated evenly with Matrigel (Sigma Aldrich, Germany). Subsequently, 1 × 104 cells in 200 µL of serum-free medium were seeded into the upper chamber. Following a 48 h incubation period, non-invading or non-migratory cells were removed. Cells were fixed with 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet (Sigma Aldrich, Germany) for 1 h. After washed by sterile water, cells were observed using an inverted fluorescence microscope (Olympus, Japan). Results were examined and documented in three random fields of view.

2.10 Detection of cell cycle and apoptosis

Cell cycle and apoptosis analyses were performed utilizing the Annexin V FITC/PI Apoptosis and Cell Cycle Kit (MultiSciences, China) following the manufacturer’s protocol. Flow cytometric analysis was performed using a Beckman instrument (USA).

2.11 In vivo experiments

Four-week-old male athymic BALB/C-nude mice were acquired from SPF Biotechnology (Beijing, China). All experimental procedures and husbandry were conducted at the Center for Animal Experiment at Wuhan University. The mice were randomly assigned and identified in two groups, with five mice per group, for the establishment of a subcutaneous tumor model. A stable UBE2Q2 knockdown cell line (Lv-shUBE2Q2) was generated using Huh-7 cells. For tumor induction, 5 × 106 cells were injected subcutaneously into the right axillary region of each mouse. Commencing on day 7 post-inoculation, mice received 100 mg/kg of PX-478 in PBS by intraperitoneal injection bi-daily. Tumor sizes were measured tri-weekly. After 3–4 weeks, the mice were euthanized, tumors were excised, and preserved in formalin for further immunohistochemical analysis.

2.12 HE and immunohistochemical staining (IHC)

Tissues intended for hematoxylin and eosin (H&E) or immunohistochemical (IHC) staining were fixed in 4% paraformaldehyde solution. These were then dehydrated, embedded in paraffin, and sectioned to produce tissue slices. Then each section was stained with H&E or with primary antibody such as UBE2Q2 antibody, HIF1α antibody or Ki67 antibody etc. Detailed information of primary antibodies used in IHC is provided in Table S3 of the supplementary materials. Imaging was conducted using an Aperio VERSA 8 automatic digital scanning and analysis system (Leica Biosystems, Germany), and the images were quantitatively analyzed with Aperio ImageScope software.

2.13 Proteomics assay

The HCC-LM3 cells transfected with UBE2Q2 plasmids constituted the experimental group, while those transfected with empty vector plasmids served as the control. Cells were harvested 48 h post-transfection. Cell pellets were lysed using a protein lysis buffer containing 7 M urea, 2 M thiourea, 4% SDS, 40 mM Tris-HCl (pH 8.5), 1 mM PMSF, and 2 mM EDTA. The lysate was sonicated for 15 min, then centrifuged at 4 °C for 20 min to collect the protein precipitate. The resulting samples were submitted in triplicate for proteomic analysis at Singleronbio (Nanjing, China). Peptides were tagged using the iTRAQ-8 kit (SCIEX), and peptide detection was conducted with TripleTOF 5600plus mass spectrometry. ProteinPilot V4.5 software was utilized for protein identification and quantification. Differential protein expression was determined based on fold change and P-value; proteins exhibiting a fold change of ≥ 1.5 (upregulated) or ≤ 0.67 (downregulated) with a P-value of ≤ 0.05 were considered significant.

2.14 Co-immunoprecipitation (Co-IP) and mass spectrometry (MS)

Huh-7 cells were grown in 10 cm diameter culture dishes and co-transfected with various plasmids for subsequent co-immunoprecipitation experiments. The cells were harvested and placed into 2 mL centrifuge tubes. Each tube was treated with 1 mL of IP buffer (comprising 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% NP-40, and 1×Protease and Phosphatase Inhibitor Cocktail) to lyse the cells for 30 min. Pre-washed Protein A/G Magnetic beads from MCE (USA) were introduced into the tubes and the mixture was agitated on a disc rotator mixer for another 30 min. Target-specific antibodies were then added to the respective tubes and incubated for over 3 h at 4 °C. After the incubation, beads were collected by centrifugation at 5,000 rpm for 2 min at 4 °C and washed 4 times with ice-cold wash buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA). The beads were resuspended with 40 µl of 2× sample buffer and boiled for 3 min. Immunoprecipitates were separated by SDS-PAGE and sent for mass spectrometry analysis (Hangzhou Cosmos Wisdom Biotechnology).

2.15 Western blot (WB)

Tissue and cellular proteins were extracted using RIPA lysis buffer supplemented with 1×PMSF proteasome inhibitor, both sourced from Beyotime (China). Protein concentrations were quantified using a BCA assay kit provided by Thermo Fisher Scientific (CA, USA). Proteins were resolved by electrophoresis on 7-10% SDS-PAGE gels and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 1 h before overnight incubation at 4℃ with primary antibodies. Following a series of washes, the membranes were incubated with the corresponding secondary antibodies for 90 min at room temperature. Then membranes were dipped in ECL Substrate Luminol solution from Bio-Rad (CA, USA). Chemiluminescence detection was performed using the Tanon-5200 imager (Shanghai, China). Comprehensive antibody details were included in Table S3 of the supplementary materials.

2.16 Glucose uptake and lactate production

Designated cells were transfected with UBE2Q2 plasmids, HIF1α plasmids, empty vector plasmids, siUBE2Q2, and siRNA NC in line with the experimental protocol, and then seeded into 96-well plates. At the 24 h mark, glucose uptake and lactate secretion were quantified using the Glucose Uptake Colorimetric Assay Kit and the L-Lactate Assay Kit, respectively, both from Sigma Aldrich (Germany) and in strict accordance with the provided manufacturer’s protocols. Absorbance readings at 412 nm were captured using an enzyme-linked immunosorbent assay reader.

2.17 Immunofluorescence (IF)

Huh-7 and HCC-LM3 cells fixed in 4% paraformaldehyde were permeabilized using 0.5% Triton X-100 for 15 min. Subsequently, cells were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature before overnight primary antibody incubation at 4 °C. After thorough washing with PBS, the cells were incubated with fluorescently labeled secondary antibodies for 1 h at room temperature within a humidified chamber. Following this, cells were mounted with DAPI to stain nuclei and visualized with an Olympus FluoView™ FV1000 confocal microscope.

2.18 Seahorse bioanalyzer

Cells transfected with the appropriate plasmids or siRNA were plated at a density of 4 × 104 cells per well in XF24 microplates (Seahorse Agilent, USA). The assay cartridge was hydrated in a CO2-free incubator at 37 °C overnight. The next day, cells were switched to a specialized assay medium before the extracellular acidification rate (ECAR) or oxygen consumption rate (OCR) was measured using a Seahorse XF24 Analyzer’s sensor probe, adhering to the manufacturer’s guidelines. For ECAR, glycolysis stress tests were conducted with Seahorse XF cell glycolysis stress test kits, and the data were processed using Wave software. For OCR, oxygen consumption tests were conducted with Seahorse XF cell mito stress test kits, and the data were processed using Wave software.

2.19 In vivo ubiquitination assays

Following 48 h post-transfection, Huh-7 cells transfected with pEZ-UBE2Q2 or vector plasmids and treated with cobalt chloride were harvested. Additionally, Huh-7 cells co-transfected with pEZ-UBE2Q2 and mutant ubiquitin plasmids—pCDEF-HA-Ub(K11) (all lysine was mutant to arginine except from wild-type lysine 11), pCDEF-HA-Ub(K48) (all lysine was mutant to arginine except from wild-type lysine 48), or pCDEF-HA-Ub(K63) (all lysine was mutant to arginine except from wild-type lysine 63)—were collected after 48 h. Cell pellets from each group were lysed in IP lysis buffer. Subsequent procedures were coherent with those previously described for co-immunoprecipitation (Co-IP).

2.20 Bioinformatics analysis

UBE2Q2 expression in paired tissue samples and associated Kaplan-Meier survival curves for hepatocellular carcinoma (HCC) patients were extracted from The Cancer Genome Atlas (TCGA) database. Additional analyses of overall survival curves and correlations with UBE2Q2 in HCC were conducted using the GEPIA platform (http://gepia.cancer-pku.cn). The data for single-gene enrichment analysis were sourced from the Gene Set Enrichment Analysis (GSEA) website (http://www.gsea-msigdb.org/gsea/index.jsp), with the analysis performed using GSEA-3.0 software.

2.21 Statistical analysis

Statistical analysis was conducted using GraphPad Prism version 8.0 (GraphPad Software) and SPSS version 22.0 (SPSS, USA). Data are expressed as mean ± standard deviation (SD). The Student’s t-test and non-parametric test were utilized for comparing differences between two groups, while two-way ANOVA was employed for analyses involving more than two groups. Correlations between UBE2Q2 expression and clinical parameters were determined using the chi-squared (χ2) test, with two-tailed P values. Significance levels for all tests were set as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, with ns denoting ‘not significant’.

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